MENU
The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.
More About: ESP | OUR CONTENT | THIS WEBSITE | WHAT'S NEW | WHAT'S HOT
ESP: PubMed Auto Bibliography 29 Oct 2024 at 01:44 Created:
CRISPR-Cas
Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.
Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion
Citations The Papers (from PubMed®)
RevDate: 2024-10-26
CmpDate: 2024-10-26
Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.
Methods in molecular biology (Clifton, N.J.), 2853:49-69.
Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.
Additional Links: PMID-39460914
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39460914,
year = {2025},
author = {Grav, LM and Rojek, JB and la Cour Karottki, KJ and Lee, JS and Kildegaard, HF},
title = {Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2853},
number = {},
pages = {49-69},
pmid = {39460914},
issn = {1940-6029},
mesh = {Animals ; CHO Cells ; *Cricetulus ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Recombinant Proteins/genetics/biosynthesis ; *Glutamate-Ammonia Ligase/genetics ; Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
CHO Cells
*Cricetulus
*CRISPR-Cas Systems
*Gene Editing/methods
*Recombinant Proteins/genetics/biosynthesis
*Glutamate-Ammonia Ligase/genetics
Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
RevDate: 2024-10-28
CmpDate: 2024-10-26
Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.
Viruses, 16(10):.
Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.
Additional Links: PMID-39459899
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39459899,
year = {2024},
author = {Kumar, A and Combe, E and Mougené, L and Zoulim, F and Testoni, B},
title = {Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.},
journal = {Viruses},
volume = {16},
number = {10},
pages = {},
pmid = {39459899},
issn = {1999-4915},
support = {ANR-23-IAHU-0008//Agence Nationale de la Recherche/ ; },
mesh = {*Hepatitis B virus/genetics ; *CRISPR-Cas Systems ; Humans ; *Hepatitis B/virology/therapy ; DNA, Viral/genetics ; DNA, Circular/genetics ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Gene Editing/methods ; Virus Replication ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Hepatitis B virus/genetics
*CRISPR-Cas Systems
Humans
*Hepatitis B/virology/therapy
DNA, Viral/genetics
DNA, Circular/genetics
Animals
Antiviral Agents/therapeutic use/pharmacology
Gene Editing/methods
Virus Replication
RevDate: 2024-10-28
CmpDate: 2024-10-28
A modified glycosylase base editor without predictable DNA off-target effects.
FEBS letters, 598(20):2557-2565.
Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBE[YE1], which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBE[YE1] showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBE[YE1] is a safe tool for gene therapy.
Additional Links: PMID-38946058
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38946058,
year = {2024},
author = {Lian, M and Chen, T and Chen, M and Peng, X and Yang, Y and Luo, X and Chi, Y and Wang, J and Tang, C and Zhou, X and Zhang, K and Qin, C and Lai, L and Zhou, J and Zou, Q},
title = {A modified glycosylase base editor without predictable DNA off-target effects.},
journal = {FEBS letters},
volume = {598},
number = {20},
pages = {2557-2565},
doi = {10.1002/1873-3468.14970},
pmid = {38946058},
issn = {1873-3468},
support = {82001974//National Natural Science Foundation of China/ ; 2023A1515011487//Natural Science Foundation of Guangdong Province/ ; 2022KQNCX095//Youth Innovation Project of Guangdong Province University/ ; JZ2022018//Guangdong Yiyang Healthcare Charity Foundation/ ; 2022YFA1105403//National Key Research and Development Program of China Stem Cell and Translational Research/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; DNA Glycosylases/metabolism/genetics ; DNA/metabolism/genetics ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBE[YE1], which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBE[YE1] showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBE[YE1] is a safe tool for gene therapy.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics
DNA Glycosylases/metabolism/genetics
DNA/metabolism/genetics
Animals
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
RevDate: 2024-10-26
CmpDate: 2024-10-26
Bovine Transcription Factor POU Class 2 Homeobox 1 (POU2F1/Oct1) Protein Promotes BoHV-1 Replication in MDBK Cells.
Viruses, 16(10):.
Bovine herpesvirus type 1 (BoHV-1) causes severe diseases in bovine species and great economic burden to the cattle industry worldwide. Due to its complex life cycle, many host factors that affect BoHV-1 replication remain to be explored. To understand the possible roles that the Oct1 cellular protein could play in this process, we first created Oct1-deficient MDBK cells using CRISPR/Cas9-mediated genome editing. Upon infection, the absence of Oct1 in MDBK cells significantly impacted BoHV-1 replication, a phenotype rescued by over-expressing the wild-type Oct1 protein in the deficient cells. We further found that the expression of all three classes of temporal genes, including essential and non-essential viral genes, were significantly reduced in Oct1 knockout MDBK cells, following both high and low multiplicity of infection. In summary, our findings confirm that the bovine Oct1 protein acts as a pro-viral factor for BoHV-1 replication by promoting its viral gene transcription in MDBK cells.
Additional Links: PMID-39459888
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39459888,
year = {2024},
author = {Rong, E and Dry, I and Dalziel, RG and Tan, WS},
title = {Bovine Transcription Factor POU Class 2 Homeobox 1 (POU2F1/Oct1) Protein Promotes BoHV-1 Replication in MDBK Cells.},
journal = {Viruses},
volume = {16},
number = {10},
pages = {},
pmid = {39459888},
issn = {1999-4915},
support = {BB/P003966/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/P013740/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/RL/230002A//UKRI-BBSRC/ ; },
mesh = {Animals ; Cattle ; *Virus Replication ; *Herpesvirus 1, Bovine/physiology/genetics ; Cell Line ; *Octamer Transcription Factor-1/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Herpesviridae Infections/virology/veterinary/metabolism/genetics ; Host-Pathogen Interactions ; Cattle Diseases/virology ; Gene Knockout Techniques ; Gene Expression Regulation, Viral ; },
abstract = {Bovine herpesvirus type 1 (BoHV-1) causes severe diseases in bovine species and great economic burden to the cattle industry worldwide. Due to its complex life cycle, many host factors that affect BoHV-1 replication remain to be explored. To understand the possible roles that the Oct1 cellular protein could play in this process, we first created Oct1-deficient MDBK cells using CRISPR/Cas9-mediated genome editing. Upon infection, the absence of Oct1 in MDBK cells significantly impacted BoHV-1 replication, a phenotype rescued by over-expressing the wild-type Oct1 protein in the deficient cells. We further found that the expression of all three classes of temporal genes, including essential and non-essential viral genes, were significantly reduced in Oct1 knockout MDBK cells, following both high and low multiplicity of infection. In summary, our findings confirm that the bovine Oct1 protein acts as a pro-viral factor for BoHV-1 replication by promoting its viral gene transcription in MDBK cells.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Cattle
*Virus Replication
*Herpesvirus 1, Bovine/physiology/genetics
Cell Line
*Octamer Transcription Factor-1/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Herpesviridae Infections/virology/veterinary/metabolism/genetics
Host-Pathogen Interactions
Cattle Diseases/virology
Gene Knockout Techniques
Gene Expression Regulation, Viral
RevDate: 2024-10-26
CmpDate: 2024-10-26
Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.
Viruses, 16(10):.
The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.
Additional Links: PMID-39459882
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39459882,
year = {2024},
author = {Wang, H and Zhou, G and Liu, H and Peng, R and Sun, T and Li, S and Chen, M and Wang, Y and Shi, Q and Xie, X},
title = {Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.},
journal = {Viruses},
volume = {16},
number = {10},
pages = {},
pmid = {39459882},
issn = {1999-4915},
support = {2021GDASYL-20210103008//GDAS' Project of Science and Technology Development/ ; 32102647//National Natural Science Foundation of China/ ; },
mesh = {*Circovirus/genetics/isolation & purification ; Animals ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/virology/diagnosis ; *CRISPR-Cas Systems ; *Circoviridae Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; },
abstract = {The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Circovirus/genetics/isolation & purification
Animals
Swine
*Nucleic Acid Amplification Techniques/methods
*Swine Diseases/virology/diagnosis
*CRISPR-Cas Systems
*Circoviridae Infections/veterinary/diagnosis/virology
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
RevDate: 2024-10-27
CmpDate: 2024-10-27
Robust aptamer-targeted CRISPR/Cas9 delivery using mesenchymal stem cell membrane -liposome hybrid: BIRC5 gene knockout against melanoma.
Nanomedicine : nanotechnology, biology, and medicine, 62:102778.
In this study, a platform was fabricated by combining a cationic lipid, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) with mesenchymal stem cell membrane (MSCM) to produce a positively charged hybrid vesicle. The prepared hybrid vesicle was used to condense BIRC5 CRISPR/Cas9 plasmid for survivin (BIRC5) gene editing. The Sgc8-c aptamer (against protein tyrosine kinase 7) was then attached to the surface of the prepared NPs through electrostatic interactions. In this regard, melanoma cancer cells (B16F0 cell line) overexpressing PTK7 receptor could be targeted. Investigations were conducted on this system to evaluate its transfection efficiency, cellular toxicity, and therapeutic performance in preclinical stage using B16F0 tumor bearing C57BL/6 J mice. The results verified the superiority of the Hybrid/ BIRC5 compared to Liposome/ BIRC5 in terms of cellular toxicity and transfection efficiency. The cells exposure to Hybrid/BIRC5 significantly enhanced cytotoxicity. Moreover, Apt-Hybrid/BIRC5 showed higher anti-proliferation activity toward PTK7-positive B16F0 cancer cells than that of the PKT7-negative CHO cell line. The active tumor targeting nanoparticles increased the cytotoxicity through down-regulation of BIRC5 expression as confirmed by Western blot analysis. In preclinical stage, Apt-Hybrid/BIRC5 showed remarkable tumor growth suppression toward B16F0 tumorized mice. Thus, our study suggested that genome editing for BIRC5 through the CRISPR/Cas9 system could provide a potentially safe approach for melanoma cancer therapy and has great potential for clinical translation.
Additional Links: PMID-39127174
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39127174,
year = {2024},
author = {Ghaemi, A and Abnous, K and Taghdisi, SM and Vakili-Azghandi, M and Ramezani, M and Alibolandi, M},
title = {Robust aptamer-targeted CRISPR/Cas9 delivery using mesenchymal stem cell membrane -liposome hybrid: BIRC5 gene knockout against melanoma.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {62},
number = {},
pages = {102778},
doi = {10.1016/j.nano.2024.102778},
pmid = {39127174},
issn = {1549-9642},
mesh = {Animals ; *Survivin/genetics/metabolism ; *Liposomes/chemistry ; *Mesenchymal Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Inbred C57BL ; Aptamers, Nucleotide/chemistry/genetics ; Gene Knockout Techniques ; Cell Line, Tumor ; Gene Editing ; Cell Membrane/metabolism ; Melanoma, Experimental/pathology/genetics ; Melanoma/genetics/pathology/therapy ; Humans ; },
abstract = {In this study, a platform was fabricated by combining a cationic lipid, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) with mesenchymal stem cell membrane (MSCM) to produce a positively charged hybrid vesicle. The prepared hybrid vesicle was used to condense BIRC5 CRISPR/Cas9 plasmid for survivin (BIRC5) gene editing. The Sgc8-c aptamer (against protein tyrosine kinase 7) was then attached to the surface of the prepared NPs through electrostatic interactions. In this regard, melanoma cancer cells (B16F0 cell line) overexpressing PTK7 receptor could be targeted. Investigations were conducted on this system to evaluate its transfection efficiency, cellular toxicity, and therapeutic performance in preclinical stage using B16F0 tumor bearing C57BL/6 J mice. The results verified the superiority of the Hybrid/ BIRC5 compared to Liposome/ BIRC5 in terms of cellular toxicity and transfection efficiency. The cells exposure to Hybrid/BIRC5 significantly enhanced cytotoxicity. Moreover, Apt-Hybrid/BIRC5 showed higher anti-proliferation activity toward PTK7-positive B16F0 cancer cells than that of the PKT7-negative CHO cell line. The active tumor targeting nanoparticles increased the cytotoxicity through down-regulation of BIRC5 expression as confirmed by Western blot analysis. In preclinical stage, Apt-Hybrid/BIRC5 showed remarkable tumor growth suppression toward B16F0 tumorized mice. Thus, our study suggested that genome editing for BIRC5 through the CRISPR/Cas9 system could provide a potentially safe approach for melanoma cancer therapy and has great potential for clinical translation.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Survivin/genetics/metabolism
*Liposomes/chemistry
*Mesenchymal Stem Cells/metabolism
*CRISPR-Cas Systems/genetics
Mice
Mice, Inbred C57BL
Aptamers, Nucleotide/chemistry/genetics
Gene Knockout Techniques
Cell Line, Tumor
Gene Editing
Cell Membrane/metabolism
Melanoma, Experimental/pathology/genetics
Melanoma/genetics/pathology/therapy
Humans
RevDate: 2024-10-26
CmpDate: 2024-10-26
A CRISPR-Cas13b System Degrades SARS-CoV and SARS-CoV-2 RNA In Vitro.
Viruses, 16(10): pii:v16101539.
In a time of climate change, population growth, and globalization, the risk of viral spread has significantly increased. The 21st century has already witnessed outbreaks of Severe Acute Respiratory Syndrome virus (SARS-CoV), Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2), Ebola virus and Influenza virus, among others. Viruses rapidly adapt and evade human immune systems, complicating the development of effective antiviral countermeasures. Consequently, the need for novel antivirals resilient to viral mutations is urgent. In this study, we developed a CRISPR-Cas13b system to target SARS-CoV-2. Interestingly, this system was also efficient against SARS-CoV, demonstrating broad-spectrum potential. Our findings highlight CRISPR-Cas13b as a promising tool for antiviral therapeutics, underscoring its potential in RNA-virus-associated pandemic responses.
Additional Links: PMID-39459873
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39459873,
year = {2024},
author = {Andersson, K and Azatyan, A and Ekenberg, M and Güçlüler, G and Sardon Puig, L and Puumalainen, M and Pramer, T and Monteil, VM and Mirazimi, A},
title = {A CRISPR-Cas13b System Degrades SARS-CoV and SARS-CoV-2 RNA In Vitro.},
journal = {Viruses},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/v16101539},
pmid = {39459873},
issn = {1999-4915},
support = {FS-2022:0010//Karolinska Institutet/ ; ID21-0086//Stiftelsen för Strategisk Forskning/ ; },
mesh = {*SARS-CoV-2/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics/metabolism ; *COVID-19/virology ; *Severe acute respiratory syndrome-related coronavirus/genetics ; Antiviral Agents/pharmacology ; },
abstract = {In a time of climate change, population growth, and globalization, the risk of viral spread has significantly increased. The 21st century has already witnessed outbreaks of Severe Acute Respiratory Syndrome virus (SARS-CoV), Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2), Ebola virus and Influenza virus, among others. Viruses rapidly adapt and evade human immune systems, complicating the development of effective antiviral countermeasures. Consequently, the need for novel antivirals resilient to viral mutations is urgent. In this study, we developed a CRISPR-Cas13b system to target SARS-CoV-2. Interestingly, this system was also efficient against SARS-CoV, demonstrating broad-spectrum potential. Our findings highlight CRISPR-Cas13b as a promising tool for antiviral therapeutics, underscoring its potential in RNA-virus-associated pandemic responses.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*SARS-CoV-2/genetics/drug effects
*CRISPR-Cas Systems
Humans
*RNA, Viral/genetics/metabolism
*COVID-19/virology
*Severe acute respiratory syndrome-related coronavirus/genetics
Antiviral Agents/pharmacology
RevDate: 2024-10-26
CmpDate: 2024-10-26
Rapid and Ultrasensitive Detection of H. aduncum via the RPA-CRISPR/Cas12a Platform.
Molecules (Basel, Switzerland), 29(20): pii:molecules29204789.
Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/μL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency.
Additional Links: PMID-39459159
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39459159,
year = {2024},
author = {Wang, X and Chen, X and Xu, T and Jin, X and Jiang, J and Guan, F},
title = {Rapid and Ultrasensitive Detection of H. aduncum via the RPA-CRISPR/Cas12a Platform.},
journal = {Molecules (Basel, Switzerland)},
volume = {29},
number = {20},
pages = {},
doi = {10.3390/molecules29204789},
pmid = {39459159},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems ; Animals ; *Anisakis/genetics/isolation & purification ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Endodeoxyribonucleases/genetics/metabolism ; Recombinases/metabolism ; Humans ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Hysterothylacium aduncum is one of six pathogens responsible for human anisakiasis. Infection with H. aduncum can cause acute abdominal symptoms and allergic reactions and is prone to misdiagnosis in clinical practice. This study aims to enhance the efficiency and accuracy of detecting H. aduncum in food ingredients. We targeted the internal transcribed spacer 1 (ITS 1) regions of Anisakis to develop a visual screening method for detecting H. aduncum using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system. By comparing the ITS 1 region sequences of eight nematode species, we designed specific primers and CRISPR RNA (crRNA). The specificity of RPA primers was screened and evaluated, and the CRISPR system was optimized. We assessed its specificity and sensitivity and performed testing on commercial samples. The results indicated that the alternative primer ADU 1 was the most effective. The final optimized concentrations were 250 nM for Cas12a, 500 nM for crRNA, and 500 nM for ssDNA. The complete test procedure was achievable within 45 min at 37 °C, with a limit of detection (LOD) of 1.27 pg/μL. The amplified product could be directly observed using a fluorescence microscope or ultraviolet lamp. Detection results for 15 Anisakis samples were entirely consistent with those obtained via Sanger sequencing, demonstrating the higher efficacy of this method for detecting and identifying H. aduncum. This visual detection method, characterized by simple operation, visual results, high sensitivity, and specificity, meets the requirements for food safety testing and enhances monitoring efficiency.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
Animals
*Anisakis/genetics/isolation & purification
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Endodeoxyribonucleases/genetics/metabolism
Recombinases/metabolism
Humans
Bacterial Proteins
CRISPR-Associated Proteins
RevDate: 2024-10-26
CmpDate: 2024-10-26
Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines.
International journal of molecular sciences, 25(20): pii:ijms252011320.
The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.
Additional Links: PMID-39457101
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39457101,
year = {2024},
author = {Liu, Z and Zhao, Y and Wu, S and Qi, S and Qiu, Y and Lian, Z},
title = {Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines.},
journal = {International journal of molecular sciences},
volume = {25},
number = {20},
pages = {},
doi = {10.3390/ijms252011320},
pmid = {39457101},
issn = {1422-0067},
support = {32072721//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Homologous Recombination ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; DNA, Circular/genetics ; Plasmids/genetics ; Genetic Vectors/genetics ; },
abstract = {The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
HEK293 Cells
*Homologous Recombination
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
DNA, Circular/genetics
Plasmids/genetics
Genetic Vectors/genetics
RevDate: 2024-10-26
CmpDate: 2024-10-26
Reporter Alleles in hiPSCs: Visual Cues on Development and Disease.
International journal of molecular sciences, 25(20): pii:ijms252011009.
Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.
Additional Links: PMID-39456792
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456792,
year = {2024},
author = {Cotta, GC and Teixeira Dos Santos, RC and Costa, GMJ and Lacerda, SMDSN},
title = {Reporter Alleles in hiPSCs: Visual Cues on Development and Disease.},
journal = {International journal of molecular sciences},
volume = {25},
number = {20},
pages = {},
doi = {10.3390/ijms252011009},
pmid = {39456792},
issn = {1422-0067},
support = {402945/2023-9//CNPq/ ; RED-00135-2//FAPEMIG/ ; RED-00079-22//FAPEMIG/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Alleles ; *Genes, Reporter ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; },
abstract = {Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Alleles
*Genes, Reporter
*Gene Editing/methods
*CRISPR-Cas Systems
Cell Differentiation/genetics
RevDate: 2024-10-26
CmpDate: 2024-10-26
Crispr-SGRU: Prediction of CRISPR/Cas9 Off-Target Activities with Mismatches and Indels Using Stacked BiGRU.
International journal of molecular sciences, 25(20): pii:ijms252010945.
CRISPR/Cas9 is a popular genome editing technology, yet its clinical application is hindered by off-target effects. Many deep learning-based methods are available for off-target prediction. However, few can predict off-target activities with insertions or deletions (indels) between single guide RNA and DNA sequence pairs. Additionally, the analysis of off-target data is challenged due to a data imbalance issue. Moreover, the prediction accuracy and interpretability remain to be improved. Here, we introduce a deep learning-based framework, named Crispr-SGRU, to predict off-target activities with mismatches and indels. This model is based on Inception and stacked BiGRU. It adopts a dice loss function to solve the inherent imbalance issue. Experimental results show our model outperforms existing methods for off-target prediction in terms of accuracy and robustness. Finally, we study the interpretability of this model through Deep SHAP and teacher-student-based knowledge distillation, and find it can provide meaningful explanations for sequence patterns regarding off-target activity.
Additional Links: PMID-39456727
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456727,
year = {2024},
author = {Zhang, G and Luo, Y and Xie, H and Dai, Z},
title = {Crispr-SGRU: Prediction of CRISPR/Cas9 Off-Target Activities with Mismatches and Indels Using Stacked BiGRU.},
journal = {International journal of molecular sciences},
volume = {25},
number = {20},
pages = {},
doi = {10.3390/ijms252010945},
pmid = {39456727},
issn = {1422-0067},
support = {2022A1515011720//Guangdong Basic and Applied Basic Research Foundation/ ; 62103249//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *INDEL Mutation ; *Gene Editing/methods ; Deep Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Base Pair Mismatch/genetics ; },
abstract = {CRISPR/Cas9 is a popular genome editing technology, yet its clinical application is hindered by off-target effects. Many deep learning-based methods are available for off-target prediction. However, few can predict off-target activities with insertions or deletions (indels) between single guide RNA and DNA sequence pairs. Additionally, the analysis of off-target data is challenged due to a data imbalance issue. Moreover, the prediction accuracy and interpretability remain to be improved. Here, we introduce a deep learning-based framework, named Crispr-SGRU, to predict off-target activities with mismatches and indels. This model is based on Inception and stacked BiGRU. It adopts a dice loss function to solve the inherent imbalance issue. Experimental results show our model outperforms existing methods for off-target prediction in terms of accuracy and robustness. Finally, we study the interpretability of this model through Deep SHAP and teacher-student-based knowledge distillation, and find it can provide meaningful explanations for sequence patterns regarding off-target activity.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*INDEL Mutation
*Gene Editing/methods
Deep Learning
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Base Pair Mismatch/genetics
RevDate: 2024-10-26
CmpDate: 2024-10-26
DeepIndel: An Interpretable Deep Learning Approach for Predicting CRISPR/Cas9-Mediated Editing Outcomes.
International journal of molecular sciences, 25(20): pii:ijms252010928.
CRISPR/Cas9 has been applied to edit the genome of various organisms, but our understanding of editing outcomes at specific sites after Cas9-mediated DNA cleavage is still limited. Several deep learning-based methods have been proposed for repair outcome prediction; however, there is still room for improvement in terms of performance regarding frameshifts and model interpretability. Here, we present DeepIndel, an end-to-end multi-label regression model for predicting repair outcomes based on the BERT-base module. We demonstrate that our model outperforms existing methods in terms of accuracy and generalizability across various metrics. Furthermore, we utilized Deep SHAP to visualize the importance of nucleotides at various positions for DNA sequence and found that mononucleotides and trinucleotides in DNA sequences surrounding the cut site play a significant role in repair outcome prediction.
Additional Links: PMID-39456711
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456711,
year = {2024},
author = {Zhang, G and Xie, H and Dai, X},
title = {DeepIndel: An Interpretable Deep Learning Approach for Predicting CRISPR/Cas9-Mediated Editing Outcomes.},
journal = {International journal of molecular sciences},
volume = {25},
number = {20},
pages = {},
doi = {10.3390/ijms252010928},
pmid = {39456711},
issn = {1422-0067},
support = {62103249//National Natural Science Foundation of China/ ; NTF20032//STU Scientific Research Foundation for Talents/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Deep Learning ; Humans ; },
abstract = {CRISPR/Cas9 has been applied to edit the genome of various organisms, but our understanding of editing outcomes at specific sites after Cas9-mediated DNA cleavage is still limited. Several deep learning-based methods have been proposed for repair outcome prediction; however, there is still room for improvement in terms of performance regarding frameshifts and model interpretability. Here, we present DeepIndel, an end-to-end multi-label regression model for predicting repair outcomes based on the BERT-base module. We demonstrate that our model outperforms existing methods in terms of accuracy and generalizability across various metrics. Furthermore, we utilized Deep SHAP to visualize the importance of nucleotides at various positions for DNA sequence and found that mononucleotides and trinucleotides in DNA sequences surrounding the cut site play a significant role in repair outcome prediction.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*Gene Editing/methods
*Deep Learning
Humans
RevDate: 2024-10-26
CmpDate: 2024-10-26
ddPCR Overcomes the CRISPR-Cas13a-Based Technique for the Detection of the BRAF p.V600E Mutation in Liquid Biopsies.
International journal of molecular sciences, 25(20): pii:ijms252010902.
The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes.
Additional Links: PMID-39456686
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456686,
year = {2024},
author = {Palacín-Aliana, I and García-Romero, N and Carrión-Navarro, J and Puig-Serra, P and Torres-Ruiz, R and Rodríguez-Perales, S and Viñal, D and González-Rumayor, V and Ayuso-Sacido, Á},
title = {ddPCR Overcomes the CRISPR-Cas13a-Based Technique for the Detection of the BRAF p.V600E Mutation in Liquid Biopsies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {20},
pages = {},
doi = {10.3390/ijms252010902},
pmid = {39456686},
issn = {1422-0067},
support = {RTC-2019-6918-1//Ministerio de Economía y Competitividad-FEDER/ ; IND2019/BMD-17222//Comunidad de Madrid/ ; PI21/01353//Instituto de Salud Carlos III and FEDER/ ; PI20/01837//Instituto de Salud Carlos III and FEDER/ ; PI21/01641//Instituto de Salud Carlos III and FEDER/ ; AECC Lab 2020, LABAE20049RODR//Asociación Española Contra el Cáncer/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins B-raf/genetics ; Liquid Biopsy/methods ; *CRISPR-Cas Systems ; Circulating Tumor DNA/genetics/blood ; Neoplasms/genetics/diagnosis ; Mutation ; Point Mutation ; Polymerase Chain Reaction/methods ; Real-Time Polymerase Chain Reaction/methods ; Reproducibility of Results ; },
abstract = {The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Proto-Oncogene Proteins B-raf/genetics
Liquid Biopsy/methods
*CRISPR-Cas Systems
Circulating Tumor DNA/genetics/blood
Neoplasms/genetics/diagnosis
Mutation
Point Mutation
Polymerase Chain Reaction/methods
Real-Time Polymerase Chain Reaction/methods
Reproducibility of Results
RevDate: 2024-10-26
Recent Advances in the CRISPR/Cas-Based Nucleic Acid Biosensor for Food Analysis: A Review.
Foods (Basel, Switzerland), 13(20): pii:foods13203222.
Food safety is a major public health issue of global concern. In recent years, the CRISPR/Cas system has shown promise in the field of molecular detection. The system has been coupled with various nucleic acid amplification methods and combined with different signal output systems to develop a new generation of CRISPR/Cas-based nucleic acid biosensor technology. This review describes the design concept of the CRISPR/Cas-based nucleic acid biosensor and its application in food analysis. A detailed overview of different CRISPR/Cas systems, signal amplification methods, and signal output strategies is provided. CRISPR/Cas-based nucleic acid biosensors have the advantages of high sensitivity, strong specificity, and timeliness, achieving fast analysis of a variety of targets, including bacteria, toxins, metal ions, pesticides, veterinary drugs, and adulteration, promoting the development of rapid food safety detection technology. At the end, we also provide our outlook for the future development of CRISPR/Cas-based nucleic acid biosensors.
Additional Links: PMID-39456285
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456285,
year = {2024},
author = {Sun, Y and Wen, T and Zhang, P and Wang, M and Xu, Y},
title = {Recent Advances in the CRISPR/Cas-Based Nucleic Acid Biosensor for Food Analysis: A Review.},
journal = {Foods (Basel, Switzerland)},
volume = {13},
number = {20},
pages = {},
doi = {10.3390/foods13203222},
pmid = {39456285},
issn = {2304-8158},
support = {Z221100007122004//Beijing Science and Technology Planning Project/ ; },
abstract = {Food safety is a major public health issue of global concern. In recent years, the CRISPR/Cas system has shown promise in the field of molecular detection. The system has been coupled with various nucleic acid amplification methods and combined with different signal output systems to develop a new generation of CRISPR/Cas-based nucleic acid biosensor technology. This review describes the design concept of the CRISPR/Cas-based nucleic acid biosensor and its application in food analysis. A detailed overview of different CRISPR/Cas systems, signal amplification methods, and signal output strategies is provided. CRISPR/Cas-based nucleic acid biosensors have the advantages of high sensitivity, strong specificity, and timeliness, achieving fast analysis of a variety of targets, including bacteria, toxins, metal ions, pesticides, veterinary drugs, and adulteration, promoting the development of rapid food safety detection technology. At the end, we also provide our outlook for the future development of CRISPR/Cas-based nucleic acid biosensors.},
}
RevDate: 2024-10-26
CmpDate: 2024-10-26
Nucleoside Analogs in ADAR Guide Strands Enable Editing at 5'-GA Sites.
Biomolecules, 14(10): pii:biom14101229.
Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.
Additional Links: PMID-39456162
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39456162,
year = {2024},
author = {Manjunath, A and Cheng, J and Campbell, KB and Jacobsen, CS and Mendoza, HG and Bierbaum, L and Jauregui-Matos, V and Doherty, EE and Fisher, AJ and Beal, PA},
title = {Nucleoside Analogs in ADAR Guide Strands Enable Editing at 5'-GA Sites.},
journal = {Biomolecules},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/biom14101229},
pmid = {39456162},
issn = {2218-273X},
support = {F31CA265135-01/CA/NCI NIH HHS/United States ; NSF GRFP 165004//National Science Foundation/ ; R01 GM149799/NH/NIH HHS/United States ; CA-D-MCB-2629-H//National Institute of Food and Agriculture/ ; },
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; Humans ; *Adenosine/analogs & derivatives/metabolism/chemistry ; Inosine/chemistry/metabolism ; Nucleosides/chemistry/metabolism ; RNA-Binding Proteins/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; RNA, Double-Stranded/chemistry/metabolism/genetics ; HEK293 Cells ; Guanosine/chemistry/metabolism/analogs & derivatives ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Adenosine Deaminase/metabolism/chemistry/genetics
*RNA Editing
Humans
*Adenosine/analogs & derivatives/metabolism/chemistry
Inosine/chemistry/metabolism
Nucleosides/chemistry/metabolism
RNA-Binding Proteins/metabolism/chemistry/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
RNA, Double-Stranded/chemistry/metabolism/genetics
HEK293 Cells
Guanosine/chemistry/metabolism/analogs & derivatives
RevDate: 2024-10-25
CmpDate: 2024-10-25
Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors.
Communications biology, 7(1):1389.
Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generate four versions of near-PAMless base editors and systematically evaluate their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We develop BEguider, a deep learning model, to accurately predict editing results for near-PAMless base editors. We also provide experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We make our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.
Additional Links: PMID-39455714
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39455714,
year = {2024},
author = {Zhou, X and Gao, J and Luo, L and Huang, C and Wu, J and Wang, X},
title = {Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {1389},
pmid = {39455714},
issn = {2399-3642},
support = {32122023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32070603//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/chemistry/metabolism ; Humans ; *Adenine/chemistry ; CRISPR-Cas Systems ; Deep Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generate four versions of near-PAMless base editors and systematically evaluate their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We develop BEguider, a deep learning model, to accurately predict editing results for near-PAMless base editors. We also provide experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We make our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*Cytosine/chemistry/metabolism
Humans
*Adenine/chemistry
CRISPR-Cas Systems
Deep Learning
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
RevDate: 2024-10-25
Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals.
Journal of pharmaceutical sciences pii:S0022-3549(24)00479-9 [Epub ahead of print].
Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.
Additional Links: PMID-39454945
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39454945,
year = {2024},
author = {Tsukamoto, T and Mizuta, H and Sakai, E and Sakurai, F and Mizugchi, H},
title = {Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals.},
journal = {Journal of pharmaceutical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.xphs.2024.10.029},
pmid = {39454945},
issn = {1520-6017},
abstract = {Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in in vitro situations differed from that in in vivo.},
}
RevDate: 2024-10-25
CmpDate: 2024-10-25
Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.
Science advances, 10(43):eadp1053.
How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ2 and σ4 domains, undergoing σ4-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.
Additional Links: PMID-39454004
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39454004,
year = {2024},
author = {Bernal-Bernal, D and Pantoja-Uceda, D and López-Alonso, JP and López-Rojo, A and López-Ruiz, JA and Galbis-Martínez, M and Ochoa-Lizarralde, B and Tascón, I and Elías-Arnanz, M and Ubarretxena-Belandia, I and Padmanabhan, S},
title = {Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.},
journal = {Science advances},
volume = {10},
number = {43},
pages = {eadp1053},
pmid = {39454004},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems ; Sigma Factor/metabolism/chemistry/genetics ; Myxococcus xanthus/metabolism ; Bacterial Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Cryoelectron Microscopy ; Bacteriophages/metabolism/genetics ; Catalytic Domain ; Protein Multimerization ; },
abstract = {How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ2 and σ4 domains, undergoing σ4-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
Sigma Factor/metabolism/chemistry/genetics
Myxococcus xanthus/metabolism
Bacterial Proteins/metabolism/chemistry/genetics
Models, Molecular
Cryoelectron Microscopy
Bacteriophages/metabolism/genetics
Catalytic Domain
Protein Multimerization
RevDate: 2024-10-25
CmpDate: 2024-10-25
CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.
Plant cell reports, 43(11):271.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.
Additional Links: PMID-39453560
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39453560,
year = {2024},
author = {Hillary, VE and Ceasar, SA},
title = {CRISPR/Cas system-mediated base editing in crops: recent developments and future prospects.},
journal = {Plant cell reports},
volume = {43},
number = {11},
pages = {271},
pmid = {39453560},
issn = {1432-203X},
support = {Under Seed Money for Faculty Minor Research//Rajagiri College of Social Sciences/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing system has altered plant research by allowing for targeted genome alteration, and they are emerging as powerful tools for evaluating plant gene function and improving crop yield. Even though CRISPR/Cas9 cleavage and subsequent repair are effective ways to precisely replace genes and change base pairs in plants, the dominance of the non-homologous end-joining pathway (NHEJ) and homology-directed repair's (HDR) poor effectiveness in plant cells have restricted their use. Base editing is gaining popularity as a potential alternative to HDR or NHEJ-mediated replacement, allowing for precise changes in the plant genome via programmed conversion of a single base to another without the need for a donor repair template or double-stranded breaks. In this review, we primarily present the mechanisms of base-editing system, including their distinct types such as DNA base editors (cytidine base editor and adenine base editor) and RNA base editors discovered so far. Next, we outline the current potential applications of the base-editing system for crop improvements. Finally, we discuss the limitations and potential future directions of the base-editing system in terms of improving crop quality. We hope that this review will enable the researcher to gain knowledge about base-editing tools and their potential applications in crop improvement.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*Gene Editing/methods
*Crops, Agricultural/genetics
Genome, Plant/genetics
Plants, Genetically Modified/genetics
RevDate: 2024-10-25
CmpDate: 2024-10-25
CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.
Functional & integrative genomics, 24(6):199.
Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.
Additional Links: PMID-39453513
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39453513,
year = {2024},
author = {Adane, M and Alamnie, G},
title = {CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.},
journal = {Functional & integrative genomics},
volume = {24},
number = {6},
pages = {199},
pmid = {39453513},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Stress, Physiological/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; Plant Breeding/methods ; },
abstract = {Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*CRISPR-Cas Systems
*Crops, Agricultural/genetics
*Stress, Physiological/genetics
Plants, Genetically Modified/genetics
Genome, Plant
Plant Breeding/methods
RevDate: 2024-10-26
CmpDate: 2024-10-25
Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges.
Biosensors, 14(10):.
The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.
Additional Links: PMID-39451674
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39451674,
year = {2024},
author = {Son, H},
title = {Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges.},
journal = {Biosensors},
volume = {14},
number = {10},
pages = {},
pmid = {39451674},
issn = {2079-6374},
support = {KGM5382423//the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA ; *RNA ; *Biosensing Techniques ; Humans ; },
abstract = {The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
*DNA
*RNA
*Biosensing Techniques
Humans
RevDate: 2024-10-25
CmpDate: 2024-10-25
Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes.
Insect biochemistry and molecular biology, 174:104192.
Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.
Additional Links: PMID-39401552
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39401552,
year = {2024},
author = {Yan, X and Zhao, Z and Feng, S and Zhang, Y and Wang, Z and Li, Z},
title = {Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes.},
journal = {Insect biochemistry and molecular biology},
volume = {174},
number = {},
pages = {104192},
doi = {10.1016/j.ibmb.2024.104192},
pmid = {39401552},
issn = {1879-0240},
mesh = {Animals ; *Spodoptera/genetics/growth & development/metabolism/physiology ; *Chitin/metabolism ; Hot Temperature ; Larva/genetics/growth & development/metabolism ; CRISPR-Cas Systems ; Insect Proteins/metabolism/genetics ; Thermotolerance ; Transcriptome ; RNA Interference ; Multiomics ; },
abstract = {Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Spodoptera/genetics/growth & development/metabolism/physiology
*Chitin/metabolism
Hot Temperature
Larva/genetics/growth & development/metabolism
CRISPR-Cas Systems
Insect Proteins/metabolism/genetics
Thermotolerance
Transcriptome
RNA Interference
Multiomics
RevDate: 2024-10-26
CmpDate: 2024-10-26
Mycobacterium tuberculosis Detection Using CRISPR Technology: An Updated Systematic Review and Meta-analysis.
Molecular diagnosis & therapy, 28(6):777-790.
BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is paramount for effective management and control of tuberculosis. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.
METHODS: A meticulous search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Quality was assessed using the Joanna Briggs Institute checklist. Data synthesis and analyses, including subgroup analyses, were performed with R v 4.3.1, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.
RESULTS: From 389 identified studies, 14 met the inclusion criteria, encompassing 2175 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.93 (95% CI 0.85-0.99) and 0.97 (95% CI 0.94-0.99), respectively. The pooled diagnostic odds ratio was 273.4379 (95% CI 103.3311-723.5794), with an area under the curve of 0.97 for the summary receiver operating characteristic (SROC) curve, denoting excellent diagnostic accuracy. Subgroup analyses illustrated variations in diagnostic metrics based on factors like CRISPR variant utilized, target gene, and pre-amplification methods. For instance, CRISPR-Cas12 exhibited a sensitivity and specificity of 0.93 (95% CI 0.78-0.98) and 0.98 (95% CI 0.93-1), respectively. Moreover, this technology showed a sensitivity of 96% and specificity of 100% in detecting resistant MTB.
CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB, with notable variances across different CRISPR variants and methodological approaches. Further studies must be conducted to optimize CRISPR's potential as a diagnostic tool for MTB in a variety of clinical and research settings.
Additional Links: PMID-39212838
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39212838,
year = {2024},
author = {Abavisani, M and Karbas Foroushan, S and Khayami, R and Sahebkar, A},
title = {Mycobacterium tuberculosis Detection Using CRISPR Technology: An Updated Systematic Review and Meta-analysis.},
journal = {Molecular diagnosis & therapy},
volume = {28},
number = {6},
pages = {777-790},
pmid = {39212838},
issn = {1179-2000},
mesh = {*Mycobacterium tuberculosis/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Tuberculosis/diagnosis/microbiology/genetics ; *Sensitivity and Specificity ; CRISPR-Cas Systems ; ROC Curve ; Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is paramount for effective management and control of tuberculosis. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.
METHODS: A meticulous search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Quality was assessed using the Joanna Briggs Institute checklist. Data synthesis and analyses, including subgroup analyses, were performed with R v 4.3.1, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.
RESULTS: From 389 identified studies, 14 met the inclusion criteria, encompassing 2175 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.93 (95% CI 0.85-0.99) and 0.97 (95% CI 0.94-0.99), respectively. The pooled diagnostic odds ratio was 273.4379 (95% CI 103.3311-723.5794), with an area under the curve of 0.97 for the summary receiver operating characteristic (SROC) curve, denoting excellent diagnostic accuracy. Subgroup analyses illustrated variations in diagnostic metrics based on factors like CRISPR variant utilized, target gene, and pre-amplification methods. For instance, CRISPR-Cas12 exhibited a sensitivity and specificity of 0.93 (95% CI 0.78-0.98) and 0.98 (95% CI 0.93-1), respectively. Moreover, this technology showed a sensitivity of 96% and specificity of 100% in detecting resistant MTB.
CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB, with notable variances across different CRISPR variants and methodological approaches. Further studies must be conducted to optimize CRISPR's potential as a diagnostic tool for MTB in a variety of clinical and research settings.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Mycobacterium tuberculosis/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Tuberculosis/diagnosis/microbiology/genetics
*Sensitivity and Specificity
CRISPR-Cas Systems
ROC Curve
Molecular Diagnostic Techniques/methods
RevDate: 2024-10-26
CmpDate: 2024-10-26
Luteinizing Hormone Receptor Mutation (LHR[N316S]) Causes Abnormal Follicular Development Revealed by Follicle Single-Cell Analysis and CRISPR/Cas9.
Interdisciplinary sciences, computational life sciences, 16(4):976-989.
Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHR[N316S] mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHR[N316S] mice exhibited reduced numbers of corpora lutea and ovulation. The LHR[N316S] mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHR[N316S] female mice with ICR wild type male mice revealed that the infertility rate of LHR[N316S] mice was 21.4% (3/14). Litter sizes from LHR[N316S] mice were smaller than those from control wild type female mice. The oocytes from LHR[N316S] mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHR[N316S] mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.
Additional Links: PMID-39150470
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39150470,
year = {2024},
author = {Zhang, C and Nie, Y and Xu, B and Mu, C and Tian, GG and Li, X and Cheng, W and Zhang, A and Li, D and Wu, J},
title = {Luteinizing Hormone Receptor Mutation (LHR[N316S]) Causes Abnormal Follicular Development Revealed by Follicle Single-Cell Analysis and CRISPR/Cas9.},
journal = {Interdisciplinary sciences, computational life sciences},
volume = {16},
number = {4},
pages = {976-989},
pmid = {39150470},
issn = {1867-1462},
support = {2022BFH02004//Key Research and Development Program of Ningxia Hui Autonomous Region/ ; ZH2018ZDA31//the interdisciplinary program of Shanghai Jiao Tong University/ ; },
mesh = {Animals ; *Receptors, LH/genetics/metabolism ; Female ; *Ovarian Follicle/metabolism/growth & development ; Mice ; *Single-Cell Analysis ; *CRISPR-Cas Systems/genetics ; Mutation ; Male ; Oocytes/metabolism ; Granulosa Cells/metabolism ; Mice, Inbred ICR ; Infertility, Female/genetics/metabolism ; },
abstract = {Abnormal interaction between granulosa cells and oocytes causes disordered development of ovarian follicles. However, the interactions between oocytes and cumulus granulosa cells (CGs), oocytes and mural granulosa cells (MGs), and CGs and MGs remain to be fully explored. Using single-cell RNA-sequencing (scRNA-seq), we determined the transcriptional profiles of oocytes, CGs and MGs in antral follicles. Analysis of scRNA-seq data revealed that CGs may regulate follicular development through the BMP15-KITL-KIT-PI3K-ARF6 pathway with elevated expression of luteinizing hormone receptor (LHR). Because internalization of the LHR is regulated by Arf6, we constructed LHR[N316S] mice by CRISPR/Cas9 to further explore mechanisms of follicular development and novel treatment strategies for female infertility. Ovaries of LHR[N316S] mice exhibited reduced numbers of corpora lutea and ovulation. The LHR[N316S] mice had a reduced rate of oocyte maturation in vitro and decreased serum progesterone levels. Mating LHR[N316S] female mice with ICR wild type male mice revealed that the infertility rate of LHR[N316S] mice was 21.4% (3/14). Litter sizes from LHR[N316S] mice were smaller than those from control wild type female mice. The oocytes from LHR[N316S] mice had an increased rate of maturation in vitro after progesterone administration in vitro. Furthermore, progesterone treated LHR[N316S] mice produced offspring numbers per litter equivalent to WT mice. These findings provide key insights into cellular interactions in ovarian follicles and provide important clues for infertility treatment.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Receptors, LH/genetics/metabolism
Female
*Ovarian Follicle/metabolism/growth & development
Mice
*Single-Cell Analysis
*CRISPR-Cas Systems/genetics
Mutation
Male
Oocytes/metabolism
Granulosa Cells/metabolism
Mice, Inbred ICR
Infertility, Female/genetics/metabolism
RevDate: 2024-10-26
CmpDate: 2024-10-26
Simultaneous multi-site editing of individual genomes using retron arrays.
Nature chemical biology, 20(11):1482-1492.
During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology-termed a multitron-for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.
Additional Links: PMID-38982310
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38982310,
year = {2024},
author = {González-Delgado, A and Lopez, SC and Rojas-Montero, M and Fishman, CB and Shipman, SL},
title = {Simultaneous multi-site editing of individual genomes using retron arrays.},
journal = {Nature chemical biology},
volume = {20},
number = {11},
pages = {1482-1492},
pmid = {38982310},
issn = {1552-4469},
support = {R21 EB031393/EB/NIBIB NIH HHS/United States ; MCB 2137692//National Science Foundation (NSF)/ ; 1DP2GM140917//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21EB031393//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; DP2 GM140917/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; DNA, Single-Stranded/genetics ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA-Directed DNA Polymerase/genetics/metabolism ; Genome/genetics ; Humans ; Metabolic Engineering/methods ; RNA, Untranslated/genetics ; },
abstract = {During recent years, the use of libraries-scale genomic manipulations scaffolded on CRISPR guide RNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, nonadjacent precise mutations remains a laborious cycle of editing, isolating an edited cell and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a noncoding RNA that contains an multicopy single-stranded DNA, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here we describe a technology-termed a multitron-for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding DNAs are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization and metabolic engineering.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
DNA, Single-Stranded/genetics
Escherichia coli/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
RNA-Directed DNA Polymerase/genetics/metabolism
Genome/genetics
Humans
Metabolic Engineering/methods
RNA, Untranslated/genetics
RevDate: 2024-10-25
Use of CRISPR Technology in Gene Editing for Tolerance to Biotic Factors in Plants: A Systematic Review.
Current issues in molecular biology, 46(10):11086-11123 pii:cimb46100659.
The objective of this systematic review (SR) was to select studies on the use of gene editing by CRISPR technology related to plant resistance to biotic stresses. We sought to evaluate articles deposited in six electronic databases, using pre-defined inclusion and exclusion criteria. This SR demonstrates that countries such as China and the United States of America stand out in studies with CRISPR/Cas. Among the most studied crops are rice, tomatoes and the model plant Arabidopsis thaliana. The most cited biotic agents include the genera, Xanthomonas, Manaporthe, Pseudomonas and Phytophthora. This SR also identifies several CRISPR/Cas-edited genes and demonstrates that plant responses to stressors are mediated by many complex signaling pathways. The Cas9 enzyme is used in most articles and Cas12 and 13 are used as additional editing tools. Furthermore, the quality of the articles included in this SR was validated by a risk of bias analysis. The information collected in this SR helps to understand the state of the art of CRISPR/Cas aimed at improving resistance to diseases and pests to understand the mechanisms involved in most host-pathogen relationships. This SR shows that the CRISPR/Cas system provides a straightforward method for rapid gene targeting, providing useful information for plant breeding programs.
Additional Links: PMID-39451539
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39451539,
year = {2024},
author = {Mascarenhas, MS and Nascimento, FDS and Rocha, AJ and Ferreira, MDS and Oliveira, WDDS and Morais Lino, LS and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP},
title = {Use of CRISPR Technology in Gene Editing for Tolerance to Biotic Factors in Plants: A Systematic Review.},
journal = {Current issues in molecular biology},
volume = {46},
number = {10},
pages = {11086-11123},
doi = {10.3390/cimb46100659},
pmid = {39451539},
issn = {1467-3045},
support = {OPP1093845//IITA - Accelerated Breeding of Better Bananas/ ; OPP1093845//Bill and Melinda Gates Foundation - Accelerated Breeding of Better Bananas/ ; },
abstract = {The objective of this systematic review (SR) was to select studies on the use of gene editing by CRISPR technology related to plant resistance to biotic stresses. We sought to evaluate articles deposited in six electronic databases, using pre-defined inclusion and exclusion criteria. This SR demonstrates that countries such as China and the United States of America stand out in studies with CRISPR/Cas. Among the most studied crops are rice, tomatoes and the model plant Arabidopsis thaliana. The most cited biotic agents include the genera, Xanthomonas, Manaporthe, Pseudomonas and Phytophthora. This SR also identifies several CRISPR/Cas-edited genes and demonstrates that plant responses to stressors are mediated by many complex signaling pathways. The Cas9 enzyme is used in most articles and Cas12 and 13 are used as additional editing tools. Furthermore, the quality of the articles included in this SR was validated by a risk of bias analysis. The information collected in this SR helps to understand the state of the art of CRISPR/Cas aimed at improving resistance to diseases and pests to understand the mechanisms involved in most host-pathogen relationships. This SR shows that the CRISPR/Cas system provides a straightforward method for rapid gene targeting, providing useful information for plant breeding programs.},
}
RevDate: 2024-10-25
CmpDate: 2024-10-25
[Correcting pathogenic mutations using prime editing: an overview].
Medecine sciences : M/S, 40(10):748-756.
Gene editing is an ever-evolving field and Prime editing technology is among the latest ones. It makes it possible to modify a gene using a Cas9 nickase that cuts a single strand of DNA. This Cas9 nickase is fused with a reverse transcriptase that copies a single guide RNA synthetized by the researcher. This technique is used on one hand to create pathogenic mutations to obtain cell or animal models with a specific mutation. On the other hand, Prime editing is also used in research to treat hereditary diseases by correcting mutations associated with a pathogenic effect. The mode of delivery of the treatment to the affected cells in living organisms constitutes a main challenge. Different methods are studied to reach the organs specific to each disease. This review article presents the latest results in the field as well as the challenges to solve to optimize the possible uses of Prime editing.
Additional Links: PMID-39450960
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39450960,
year = {2024},
author = {Bouchard, C and Godbout, K and Tremblay, JP},
title = {[Correcting pathogenic mutations using prime editing: an overview].},
journal = {Medecine sciences : M/S},
volume = {40},
number = {10},
pages = {748-756},
doi = {10.1051/medsci/2024109},
pmid = {39450960},
issn = {1958-5381},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *Mutation ; *CRISPR-Cas Systems ; Genetic Therapy/methods/trends ; Genetic Diseases, Inborn/genetics/therapy ; },
abstract = {Gene editing is an ever-evolving field and Prime editing technology is among the latest ones. It makes it possible to modify a gene using a Cas9 nickase that cuts a single strand of DNA. This Cas9 nickase is fused with a reverse transcriptase that copies a single guide RNA synthetized by the researcher. This technique is used on one hand to create pathogenic mutations to obtain cell or animal models with a specific mutation. On the other hand, Prime editing is also used in research to treat hereditary diseases by correcting mutations associated with a pathogenic effect. The mode of delivery of the treatment to the affected cells in living organisms constitutes a main challenge. Different methods are studied to reach the organs specific to each disease. This review article presents the latest results in the field as well as the challenges to solve to optimize the possible uses of Prime editing.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Gene Editing/methods
Animals
*Mutation
*CRISPR-Cas Systems
Genetic Therapy/methods/trends
Genetic Diseases, Inborn/genetics/therapy
RevDate: 2024-10-25
CmpDate: 2024-10-25
Standardizing CRISPR-Cas13 knockdown technique to investigate the role of cdh2 gene in pituitary development through growth hormone expression and transcription factors.
Frontiers in endocrinology, 15:1466638.
INTRODUCTION: Congenital hypopituitarism (CH) is characterized by the deficiency of pituitary hormones. Among CH patients, 85% lack a molecular diagnosis. Whole Exome Sequencing (WES) identified a homozygous variant (c.865G>A, p.Val289Ile) in the CDH2 gene, responsible for N-Cadherin production, crucial for cell-cell adhesion. Predicted to be likely pathogenic, the variant was found in a patient deficient in GH, TSH, ACTH, and LH/FSH. Its impact on cell adhesion was confirmed in L1 fibroblast cell lines.
OBJECTIVE: Create a cdh2 knockdown in zebrafish for investigating its role in pituitary development through growth hormone and transcription factors expression.
METHODS: Utilized pET28B-RfxCas13d-His plasmid for Cas13 mRNA production via in vitro transcription, guiding Cas13 to cdh2 with three RNAs. Injected the complex into single-cell embryos for analysis up to 96 hpf. Assessed gene expression of cdh2, prop1, pit1, and gh1 using RT-qPCR. Evaluated cdh2 protein expression through the western blot technique.
RESULTS: Knockdown animals displayed developmental delay. The cdh2 expression decreased by 75% within 24 hours, rebounded by 48 hours, and reached wild-type levels by 96 hpf. gh1 expression decreased at 48h but increased by 96 hpf, aligning with WT. No significant differences in prop1 and pit1 expression were observed.
CONCLUSION: Our findings underscore cdh2's role in pituitary development and hormonal regulation, offering insights for developmental biology research.
Additional Links: PMID-39449741
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39449741,
year = {2024},
author = {Ventura Fernandes, BH and Junqueira, MS and MacRae, C and Silveira de Carvalho, LR},
title = {Standardizing CRISPR-Cas13 knockdown technique to investigate the role of cdh2 gene in pituitary development through growth hormone expression and transcription factors.},
journal = {Frontiers in endocrinology},
volume = {15},
number = {},
pages = {1466638},
pmid = {39449741},
issn = {1664-2392},
mesh = {Animals ; *Zebrafish/genetics ; *Cadherins/genetics/metabolism ; *Pituitary Gland/metabolism ; *Transcription Factors/genetics/metabolism ; *Gene Knockdown Techniques ; *CRISPR-Cas Systems ; Humans ; Growth Hormone/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Hypopituitarism/genetics/metabolism ; },
abstract = {INTRODUCTION: Congenital hypopituitarism (CH) is characterized by the deficiency of pituitary hormones. Among CH patients, 85% lack a molecular diagnosis. Whole Exome Sequencing (WES) identified a homozygous variant (c.865G>A, p.Val289Ile) in the CDH2 gene, responsible for N-Cadherin production, crucial for cell-cell adhesion. Predicted to be likely pathogenic, the variant was found in a patient deficient in GH, TSH, ACTH, and LH/FSH. Its impact on cell adhesion was confirmed in L1 fibroblast cell lines.
OBJECTIVE: Create a cdh2 knockdown in zebrafish for investigating its role in pituitary development through growth hormone and transcription factors expression.
METHODS: Utilized pET28B-RfxCas13d-His plasmid for Cas13 mRNA production via in vitro transcription, guiding Cas13 to cdh2 with three RNAs. Injected the complex into single-cell embryos for analysis up to 96 hpf. Assessed gene expression of cdh2, prop1, pit1, and gh1 using RT-qPCR. Evaluated cdh2 protein expression through the western blot technique.
RESULTS: Knockdown animals displayed developmental delay. The cdh2 expression decreased by 75% within 24 hours, rebounded by 48 hours, and reached wild-type levels by 96 hpf. gh1 expression decreased at 48h but increased by 96 hpf, aligning with WT. No significant differences in prop1 and pit1 expression were observed.
CONCLUSION: Our findings underscore cdh2's role in pituitary development and hormonal regulation, offering insights for developmental biology research.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Zebrafish/genetics
*Cadherins/genetics/metabolism
*Pituitary Gland/metabolism
*Transcription Factors/genetics/metabolism
*Gene Knockdown Techniques
*CRISPR-Cas Systems
Humans
Growth Hormone/genetics/metabolism
Zebrafish Proteins/genetics/metabolism
Gene Expression Regulation, Developmental
Hypopituitarism/genetics/metabolism
RevDate: 2024-10-25
CmpDate: 2024-10-25
Plant conservation in the age of genome editing: opportunities and challenges.
Genome biology, 25(1):279.
Numerous plant taxa are threatened by habitat destruction or overexploitation. To overcome these threats, new methods are urgently needed for rescuing threatened and endangered plant species. Here, we review the genetic consequences of threats to species populations. We highlight potential advantages of genome editing for mitigating negative effects caused by new pathogens and pests or climate change where other approaches have failed. We propose solutions to protect threatened plants using genome editing technology unless absolutely necessary. We further discuss the challenges associated with genome editing in plant conservation to mitigate the decline of plant diversity.
Additional Links: PMID-39449103
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39449103,
year = {2024},
author = {Yin, K and Chung, MY and Lan, B and Du, FK and Chung, MG},
title = {Plant conservation in the age of genome editing: opportunities and challenges.},
journal = {Genome biology},
volume = {25},
number = {1},
pages = {279},
pmid = {39449103},
issn = {1474-760X},
support = {42071060//National Natural Science Foundation of China/ ; 2021ZY80//Fundamental Research Funds for the Central Universities/ ; 2022JBGS0020//Science and Technology Innovation of Inner Mongolia Autonomous Region/ ; KFJ-STS-ZDTP-2022-001//Special Program for the Institute of National Parks/ ; NIBR202305202//National Institute of Biological Resources (NIBR)/ ; },
mesh = {*Gene Editing ; *Plants/genetics ; *Genome, Plant ; *Endangered Species ; *Conservation of Natural Resources ; Climate Change ; CRISPR-Cas Systems ; },
abstract = {Numerous plant taxa are threatened by habitat destruction or overexploitation. To overcome these threats, new methods are urgently needed for rescuing threatened and endangered plant species. Here, we review the genetic consequences of threats to species populations. We highlight potential advantages of genome editing for mitigating negative effects caused by new pathogens and pests or climate change where other approaches have failed. We propose solutions to protect threatened plants using genome editing technology unless absolutely necessary. We further discuss the challenges associated with genome editing in plant conservation to mitigate the decline of plant diversity.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing
*Plants/genetics
*Genome, Plant
*Endangered Species
*Conservation of Natural Resources
Climate Change
CRISPR-Cas Systems
RevDate: 2024-10-25
CmpDate: 2024-10-25
Selective RNA pseudouridinylation in situ by circular gRNAs in designer organelles.
Nature communications, 15(1):9177.
RNA modifications play a pivotal role in the regulation of RNA chemistry within cells. Several technologies have been developed with the goal of using RNA modifications to regulate cellular biochemistry selectively, but achieving selective and precise modifications remains a challenge. Here, we show that by using designer organelles, we can modify mRNA with pseudouridine in a highly selective and guide-RNA-dependent manner. We use designer organelles inspired by concepts of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we use circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies introduce spatial engineering through optimized RNA editing organelles (OREO) as a complementary tool for targeted RNA modification, providing new avenues to enhance RNA modification specificity.
Additional Links: PMID-39448590
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39448590,
year = {2024},
author = {Schartel, L and Jann, C and Wierczeiko, A and Butto, T and Mündnich, S and Marchand, V and Motorin, Y and Helm, M and Gerber, S and Lemke, EA},
title = {Selective RNA pseudouridinylation in situ by circular gRNAs in designer organelles.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {9177},
pmid = {39448590},
issn = {2041-1723},
mesh = {*Pseudouridine/metabolism ; Humans ; *Organelles/metabolism ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *RNA, Messenger/metabolism/genetics ; HEK293 Cells ; RNA, Circular/metabolism/genetics ; RNA/metabolism/genetics ; HeLa Cells ; },
abstract = {RNA modifications play a pivotal role in the regulation of RNA chemistry within cells. Several technologies have been developed with the goal of using RNA modifications to regulate cellular biochemistry selectively, but achieving selective and precise modifications remains a challenge. Here, we show that by using designer organelles, we can modify mRNA with pseudouridine in a highly selective and guide-RNA-dependent manner. We use designer organelles inspired by concepts of phase separation, a central tenet in developing artificial membraneless organelles in living mammalian cells. In addition, we use circular guide RNAs to markedly enhance the effectiveness of targeted pseudouridinylation. Our studies introduce spatial engineering through optimized RNA editing organelles (OREO) as a complementary tool for targeted RNA modification, providing new avenues to enhance RNA modification specificity.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Pseudouridine/metabolism
Humans
*Organelles/metabolism
*RNA Editing
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*RNA, Messenger/metabolism/genetics
HEK293 Cells
RNA, Circular/metabolism/genetics
RNA/metabolism/genetics
HeLa Cells
RevDate: 2024-10-24
CmpDate: 2024-10-24
Enhancement of fermentation traits in industrial Baker's yeast for low or high sugar environments.
Food microbiology, 125:104643.
Saccharomyces cerevisiae SPC-SNU 70-1 is a commercial diploid baking yeast strain valued for its excellent bread-making qualities, including superior leavening capabilities and the production of flavor-enhancing volatile organic acids. Despite its benefits, this strain faces challenges in fermenting both lean (low-sugar) and sweet (high-sugar) doughs. To address these issues, we employed the CRISPR/Cas9 genome editing system to modify genes without leaving any genetic scars. For lean doughs, we enhanced the yeast's ability to utilize maltose over glucose by deleting a gene involved in glucose repression. For sweet doughs, we increased glycerol production by overexpressing glycerol biosynthetic genes and optimizing redox balance, thereby improving the tolerence to osmotic stress during fermentation. Additionally, the glycerol-overproducing strain demonstrated enhanced freeze tolerance, and bread made from this strain exhibited improved storage properties. This study demonstrates the feasibility and benefits of using engineered yeast strains, created solely by editing their own genes without introducing foreign genes, to enhance bread making.
Additional Links: PMID-39448153
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39448153,
year = {2025},
author = {Cha, S and Cho, K and Lim, N and Oh, H and Choi, E and Shim, S and Lee, SH and Hahn, JS},
title = {Enhancement of fermentation traits in industrial Baker's yeast for low or high sugar environments.},
journal = {Food microbiology},
volume = {125},
number = {},
pages = {104643},
doi = {10.1016/j.fm.2024.104643},
pmid = {39448153},
issn = {1095-9998},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Fermentation ; *Bread/microbiology ; *Glycerol/metabolism ; Gene Editing ; Glucose/metabolism ; CRISPR-Cas Systems ; Maltose/metabolism ; Sugars/metabolism ; Industrial Microbiology ; },
abstract = {Saccharomyces cerevisiae SPC-SNU 70-1 is a commercial diploid baking yeast strain valued for its excellent bread-making qualities, including superior leavening capabilities and the production of flavor-enhancing volatile organic acids. Despite its benefits, this strain faces challenges in fermenting both lean (low-sugar) and sweet (high-sugar) doughs. To address these issues, we employed the CRISPR/Cas9 genome editing system to modify genes without leaving any genetic scars. For lean doughs, we enhanced the yeast's ability to utilize maltose over glucose by deleting a gene involved in glucose repression. For sweet doughs, we increased glycerol production by overexpressing glycerol biosynthetic genes and optimizing redox balance, thereby improving the tolerence to osmotic stress during fermentation. Additionally, the glycerol-overproducing strain demonstrated enhanced freeze tolerance, and bread made from this strain exhibited improved storage properties. This study demonstrates the feasibility and benefits of using engineered yeast strains, created solely by editing their own genes without introducing foreign genes, to enhance bread making.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Saccharomyces cerevisiae/genetics/metabolism
*Fermentation
*Bread/microbiology
*Glycerol/metabolism
Gene Editing
Glucose/metabolism
CRISPR-Cas Systems
Maltose/metabolism
Sugars/metabolism
Industrial Microbiology
RevDate: 2024-10-25
CmpDate: 2024-10-24
Impact of CRISPR in cancer drug discovery.
Science (New York, N.Y.), 386(6720):378-379.
Precision gene editing enables massively parallel identification of cancer-promoting genes.
Additional Links: PMID-39446947
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39446947,
year = {2024},
author = {Moffat, J and Komor, AC and Lum, L},
title = {Impact of CRISPR in cancer drug discovery.},
journal = {Science (New York, N.Y.)},
volume = {386},
number = {6720},
pages = {378-379},
doi = {10.1126/science.adi6884},
pmid = {39446947},
issn = {1095-9203},
mesh = {Animals ; Humans ; *Antineoplastic Agents/therapeutic use/pharmacology ; *CRISPR-Cas Systems ; *Drug Discovery ; *Gene Editing/methods ; *Neoplasms/genetics/drug therapy ; },
abstract = {Precision gene editing enables massively parallel identification of cancer-promoting genes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Humans
*Antineoplastic Agents/therapeutic use/pharmacology
*CRISPR-Cas Systems
*Drug Discovery
*Gene Editing/methods
*Neoplasms/genetics/drug therapy
RevDate: 2024-10-24
Nanotechnology and CRISPR/Cas-Mediated Gene Therapy Strategies: Potential Role for Treating Genetic Disorders.
Molecular biotechnology [Epub ahead of print].
Gene therapy has made substantial progress in the treatment of the genetic diseases, focussing on the reduction of characteristics of recessive/dominant disorders, as well as various cancers. Extensive research has been conducted in the past few decades to investigate the application of nanotechnology and CRISPR/Cas technology in gene therapy. Nanotechnology due to attributes such has targeted drug delivery, controlled release, scalability and low toxicity has gained attention of the medical world. CRISPR/Cas9 system is considered as an impactful genome editing tool in the area of next-generation therapeutics and molecular diagnostics. CRISPR technology emphasises on gene editing, gene regulation modulation, and formulation of defined genetic changes. Its applications in treatment of the genetic disorders are extended beyond traditional therapies. These techniques are being explored as treatment of several genetic disorders including Duchenne muscular dystrophy, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and Huntington disease. Despite considerable therapeutic potential of gene therapy, several obstacles must be addressed before it can be widely adopted in clinical practice, particularly in terms of ensuring safety and effectiveness. As research advances in this captivating field, these therapies will become the primary treatments and will have significant beneficial effects on the lives of patients with genetic disorders.
Additional Links: PMID-39446301
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39446301,
year = {2024},
author = {Kaur, G and Arora, J and Sodhi, AS and Bhatia, S and Batra, N},
title = {Nanotechnology and CRISPR/Cas-Mediated Gene Therapy Strategies: Potential Role for Treating Genetic Disorders.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {39446301},
issn = {1559-0305},
abstract = {Gene therapy has made substantial progress in the treatment of the genetic diseases, focussing on the reduction of characteristics of recessive/dominant disorders, as well as various cancers. Extensive research has been conducted in the past few decades to investigate the application of nanotechnology and CRISPR/Cas technology in gene therapy. Nanotechnology due to attributes such has targeted drug delivery, controlled release, scalability and low toxicity has gained attention of the medical world. CRISPR/Cas9 system is considered as an impactful genome editing tool in the area of next-generation therapeutics and molecular diagnostics. CRISPR technology emphasises on gene editing, gene regulation modulation, and formulation of defined genetic changes. Its applications in treatment of the genetic disorders are extended beyond traditional therapies. These techniques are being explored as treatment of several genetic disorders including Duchenne muscular dystrophy, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and Huntington disease. Despite considerable therapeutic potential of gene therapy, several obstacles must be addressed before it can be widely adopted in clinical practice, particularly in terms of ensuring safety and effectiveness. As research advances in this captivating field, these therapies will become the primary treatments and will have significant beneficial effects on the lives of patients with genetic disorders.},
}
RevDate: 2024-10-24
CmpDate: 2024-10-24
Generation of CRISPR/Cas9 modified human iPSC line with correction of heterozygous mutation in exon 6 of the CaSR gene.
Human cell, 38(1):6.
The calcium-sensing receptor (CaSR) gene encodes a cell membrane G protein-coupled receptor (GPCR) which has a key role in maintaining the extracellular Ca[2+] homeostasis. We aimed at correcting the compound heterozygous mutation in the 6th [c.1656delA, p.I554SfsX73] and 7th [c.2217 T > A, p.C739X] exons of the CASR gene which the original patient-derived iPSC line had. The mutation is associated with neonatal severe primary hyperparathyroidism of the patient. We generated and characterized a CRISP/Cas9-edited hiPSC line with the restored sequence in the sixth exon of the CASR gene, bearing only heterozygous mutation in the 7th exon. The results showed that the new genetically modified cell line has karyotype without abnormalities, typical hiPSCs morphology, characteristic expression of pluripotency markers, and ability to develop into three germ layers, and differentiates in chondrogenic, adipogenic, osteogenic directions. This new cell line will complement the existing pool of CaSR-mutated cell lines, a valuable resource for in-depth understanding of neonatal severe primary hyperparathyroidism. This will allow further exploration of the application of pharmacological drugs in the context of personalized medicine to correct Ca-homeostasis disorders.
Additional Links: PMID-39446198
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39446198,
year = {2024},
author = {Semenova, PI and Panova, AV and Sopova, JV and Krasnova, OA and Turilova, VI and Yakovleva, TK and Kulikova, KS and Petrova, DA and Kiselev, SL and Neganova, IE},
title = {Generation of CRISPR/Cas9 modified human iPSC line with correction of heterozygous mutation in exon 6 of the CaSR gene.},
journal = {Human cell},
volume = {38},
number = {1},
pages = {6},
pmid = {39446198},
issn = {1749-0774},
support = {075-15-2021-1075//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Receptors, Calcium-Sensing/genetics ; Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Exons/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; *Heterozygote ; Cell Line ; Cell Differentiation/genetics ; Gene Editing/methods ; Hyperparathyroidism, Primary/genetics/therapy ; Precision Medicine ; Infant, Newborn ; Infant, Newborn, Diseases ; },
abstract = {The calcium-sensing receptor (CaSR) gene encodes a cell membrane G protein-coupled receptor (GPCR) which has a key role in maintaining the extracellular Ca[2+] homeostasis. We aimed at correcting the compound heterozygous mutation in the 6th [c.1656delA, p.I554SfsX73] and 7th [c.2217 T > A, p.C739X] exons of the CASR gene which the original patient-derived iPSC line had. The mutation is associated with neonatal severe primary hyperparathyroidism of the patient. We generated and characterized a CRISP/Cas9-edited hiPSC line with the restored sequence in the sixth exon of the CASR gene, bearing only heterozygous mutation in the 7th exon. The results showed that the new genetically modified cell line has karyotype without abnormalities, typical hiPSCs morphology, characteristic expression of pluripotency markers, and ability to develop into three germ layers, and differentiates in chondrogenic, adipogenic, osteogenic directions. This new cell line will complement the existing pool of CaSR-mutated cell lines, a valuable resource for in-depth understanding of neonatal severe primary hyperparathyroidism. This will allow further exploration of the application of pharmacological drugs in the context of personalized medicine to correct Ca-homeostasis disorders.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Receptors, Calcium-Sensing/genetics
Humans
*Induced Pluripotent Stem Cells/metabolism
*Exons/genetics
*CRISPR-Cas Systems/genetics
*Mutation/genetics
*Heterozygote
Cell Line
Cell Differentiation/genetics
Gene Editing/methods
Hyperparathyroidism, Primary/genetics/therapy
Precision Medicine
Infant, Newborn
Infant, Newborn, Diseases
RevDate: 2024-10-24
CmpDate: 2024-10-24
The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus.
Microbial pathogenesis, 196:106959.
Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/μl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.
Additional Links: PMID-39303955
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39303955,
year = {2024},
author = {Tang, X and Li, W and Wang, H and Sheng, X and Xing, J and Chi, H and Guo, M and Zhan, W},
title = {The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus.},
journal = {Microbial pathogenesis},
volume = {196},
number = {},
pages = {106959},
doi = {10.1016/j.micpath.2024.106959},
pmid = {39303955},
issn = {1096-1208},
mesh = {*CRISPR-Cas Systems ; Animals ; *Sensitivity and Specificity ; *Fish Diseases/virology/diagnosis ; Nucleic Acid Amplification Techniques/methods ; Rhabdoviridae Infections/veterinary/diagnosis/virology ; Rhabdoviridae/genetics/isolation & purification ; Fishes/virology ; Reverse Transcription ; CRISPR-Associated Proteins/genetics ; Recombinases/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/μl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems
Animals
*Sensitivity and Specificity
*Fish Diseases/virology/diagnosis
Nucleic Acid Amplification Techniques/methods
Rhabdoviridae Infections/veterinary/diagnosis/virology
Rhabdoviridae/genetics/isolation & purification
Fishes/virology
Reverse Transcription
CRISPR-Associated Proteins/genetics
Recombinases/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases
RevDate: 2024-10-25
CmpDate: 2024-10-25
Protein profiling of zebrafish embryos unmasks regulatory layers during early embryogenesis.
Cell reports, 43(10):114769.
The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data show that, unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first 2 h post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a complementary approach to unraveling maternal factor function during embryonic development.
Additional Links: PMID-39302832
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39302832,
year = {2024},
author = {da Silva Pescador, G and Baia Amaral, D and Varberg, JM and Zhang, Y and Hao, Y and Florens, L and Bazzini, AA},
title = {Protein profiling of zebrafish embryos unmasks regulatory layers during early embryogenesis.},
journal = {Cell reports},
volume = {43},
number = {10},
pages = {114769},
doi = {10.1016/j.celrep.2024.114769},
pmid = {39302832},
issn = {2211-1247},
support = {R21 OD034161/OD/NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/embryology/metabolism/genetics ; *Embryonic Development/genetics ; *Zebrafish Proteins/metabolism/genetics ; *Gene Expression Regulation, Developmental ; Embryo, Nonmammalian/metabolism ; RNA, Messenger/metabolism/genetics ; Zygote/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data show that, unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first 2 h post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a complementary approach to unraveling maternal factor function during embryonic development.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Zebrafish/embryology/metabolism/genetics
*Embryonic Development/genetics
*Zebrafish Proteins/metabolism/genetics
*Gene Expression Regulation, Developmental
Embryo, Nonmammalian/metabolism
RNA, Messenger/metabolism/genetics
Zygote/metabolism
CRISPR-Cas Systems/genetics
RevDate: 2024-10-24
Conformational change as a mechanism for toxin activation in bacterial toxin-antitoxin systems.
Journal of virology [Epub ahead of print].
Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics.
Additional Links: PMID-39445801
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39445801,
year = {2024},
author = {Sanchez-Torres, V and Hwang, H-J and Wood, TK},
title = {Conformational change as a mechanism for toxin activation in bacterial toxin-antitoxin systems.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0151324},
doi = {10.1128/jvi.01513-24},
pmid = {39445801},
issn = {1098-5514},
abstract = {Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics.},
}
RevDate: 2024-10-24
CRISPR/Cas12a assay for amol level microRNA by combining enzyme-free amplification and single particle analysis.
Chemical communications (Cambridge, England) [Epub ahead of print].
CRISPR/Cas systems are increasingly utilized for sensitive miRNA detection through enzyme-based pre-amplification. To address challenges such as high costs, non-specific amplification, and interference from primer residues in pre-amplification strategies, herein a dual amplification CRISPR miRNA assay was developed by combining enzyme-free HCR with single-particle analysis. Attomolar detection limits, excellent selectivity, and practicability were achieved by applying this method.
Additional Links: PMID-39445763
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39445763,
year = {2024},
author = {Zhang, C and Zhao, X and Chen, X and Lin, X and Huang, Z and Hu, J and Liu, R and Lv, Y},
title = {CRISPR/Cas12a assay for amol level microRNA by combining enzyme-free amplification and single particle analysis.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4cc04534c},
pmid = {39445763},
issn = {1364-548X},
abstract = {CRISPR/Cas systems are increasingly utilized for sensitive miRNA detection through enzyme-based pre-amplification. To address challenges such as high costs, non-specific amplification, and interference from primer residues in pre-amplification strategies, herein a dual amplification CRISPR miRNA assay was developed by combining enzyme-free HCR with single-particle analysis. Attomolar detection limits, excellent selectivity, and practicability were achieved by applying this method.},
}
RevDate: 2024-10-24
An Optimized CRISPR/Cas12a Assay to Facilitate the BRAF V600E Mutation Detection.
Journal of clinical laboratory analysis [Epub ahead of print].
BACKGROUND: Accurate detection of the BRAF V600E (1799T > A) mutation status can significantly contribute to selecting an optimal therapeutic strategy for diverse cancer types. CRISPR-based diagnostic platforms exhibit simple programming, cost-effectiveness, high sensitivity, and high specificity in detecting target sequences. The goal of this study is to develop a simple BRAF V600E mutation detection method.
METHODS: We combined the CRISPR/Cas12a system with recombinase polymerase amplification (RPA). Subsequently, several parameters related to CRISPR/Cas12a reaction efficiency were evaluated. Then, we conducted a comparative analysis of three distinct approaches toward identifying BRAF V600E mutations in the clinical samples.
RESULTS: Our data suggest that CRISPR/Cas detection is considerably responsive to variations in buffer conditions. Magnesium acetate (MgOAc) demonstrated superior performance compared to all other examined additive salts. It was observed using 150 nM guide RNA (gRNA) in an optimized reaction buffer containing 14 mM MgOAc, coupled with a reduction in the volumes of PCR and RPA products to 1 μL and 3 μL, respectively, resulted in an enhanced sensitivity. Detection time was decreased to 75 min with a 2% limit of detection (LOD), as evidenced by the results obtained from the blue light illuminator. The CRISPR/Cas12a assay confirmed the real-time PCR results in 31 of 32 clinical samples to identify the BRAF V600E mutation status, while Sanger sequencing detected BRAF V600E mutations with lower sensitivity.
CONCLUSION: We propose a potential diagnostic approach that is facile, fast, and affordable with high fidelity. This method can detect BRAF V600E mutation with a 2% LOD without the need for a thermocycler.
Additional Links: PMID-39445676
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39445676,
year = {2024},
author = {Etemadzadeh, A and Salehipour, P and Motlagh, FM and Khalifeh, M and Asadbeigi, A and Tabrizi, M and Shirkouhi, R and Modarressi, MH},
title = {An Optimized CRISPR/Cas12a Assay to Facilitate the BRAF V600E Mutation Detection.},
journal = {Journal of clinical laboratory analysis},
volume = {},
number = {},
pages = {e25101},
doi = {10.1002/jcla.25101},
pmid = {39445676},
issn = {1098-2825},
support = {1401-3-366-57150//Tehran University of Medical Sciences and Health Services/ ; },
abstract = {BACKGROUND: Accurate detection of the BRAF V600E (1799T > A) mutation status can significantly contribute to selecting an optimal therapeutic strategy for diverse cancer types. CRISPR-based diagnostic platforms exhibit simple programming, cost-effectiveness, high sensitivity, and high specificity in detecting target sequences. The goal of this study is to develop a simple BRAF V600E mutation detection method.
METHODS: We combined the CRISPR/Cas12a system with recombinase polymerase amplification (RPA). Subsequently, several parameters related to CRISPR/Cas12a reaction efficiency were evaluated. Then, we conducted a comparative analysis of three distinct approaches toward identifying BRAF V600E mutations in the clinical samples.
RESULTS: Our data suggest that CRISPR/Cas detection is considerably responsive to variations in buffer conditions. Magnesium acetate (MgOAc) demonstrated superior performance compared to all other examined additive salts. It was observed using 150 nM guide RNA (gRNA) in an optimized reaction buffer containing 14 mM MgOAc, coupled with a reduction in the volumes of PCR and RPA products to 1 μL and 3 μL, respectively, resulted in an enhanced sensitivity. Detection time was decreased to 75 min with a 2% limit of detection (LOD), as evidenced by the results obtained from the blue light illuminator. The CRISPR/Cas12a assay confirmed the real-time PCR results in 31 of 32 clinical samples to identify the BRAF V600E mutation status, while Sanger sequencing detected BRAF V600E mutations with lower sensitivity.
CONCLUSION: We propose a potential diagnostic approach that is facile, fast, and affordable with high fidelity. This method can detect BRAF V600E mutation with a 2% LOD without the need for a thermocycler.},
}
RevDate: 2024-10-24
CmpDate: 2024-10-24
Identification of porcine PARP11 as a restricted factor for pseudorabies virus.
Frontiers in cellular and infection microbiology, 14:1414827.
INTRODUCTION: PRV infection in swine can cause devastating disease and pose a potential threat to humans. Advancing the interplay between PRV and host is essential to elucidate the pathogenic mechanism of PRV and identify novel anti-PRV targets.
METHODS: PARP11-KO PK-15 cells were firstly constructed by CRISPR/Cas9 technology. Next, the effect of PARP11-KO on PRV infection was determined by RT-qPCR, TCID50 assay, RNA-seq, and western blot.
RESULTS AND DISCUSSION: In this study, we identified PARP11 as a host factor that can significantly affect PRV infection. Inhibition of PARP11 and knockout of PARP11 can significantly promoted PRV infection. Subsequently, we further found that PARP11 knockout upregulated the transcription of NXF1 and CRM1, resulting in enhanced transcription of viral genes. Furthermore, we also found that PARP11 knockout could activate the autophagy pathway and suppress the mTOR pathway during PRV infection. These findings could provide insight into the mechanism in which PARP11 participated during PRV infection and offer a potential target to develop anti-PRV therapies.
Additional Links: PMID-39445214
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39445214,
year = {2024},
author = {Qi, C and Zhao, D and Wang, X and Hu, L and Wang, Y and Wu, H and Li, F and Zhou, J and Zhang, T and Qi, A and Huo, Y and Tu, Q and Zhong, S and Yuan, H and Lv, D and Yan, S and Ouyang, H and Pang, D and Xie, Z},
title = {Identification of porcine PARP11 as a restricted factor for pseudorabies virus.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1414827},
pmid = {39445214},
issn = {2235-2988},
mesh = {Animals ; Swine ; *Herpesvirus 1, Suid/genetics ; Cell Line ; *Host-Pathogen Interactions ; *Gene Knockout Techniques ; *Poly(ADP-ribose) Polymerases/metabolism/genetics ; CRISPR-Cas Systems ; Autophagy ; Pseudorabies/virology ; Virus Replication ; Swine Diseases/virology ; TOR Serine-Threonine Kinases/metabolism ; },
abstract = {INTRODUCTION: PRV infection in swine can cause devastating disease and pose a potential threat to humans. Advancing the interplay between PRV and host is essential to elucidate the pathogenic mechanism of PRV and identify novel anti-PRV targets.
METHODS: PARP11-KO PK-15 cells were firstly constructed by CRISPR/Cas9 technology. Next, the effect of PARP11-KO on PRV infection was determined by RT-qPCR, TCID50 assay, RNA-seq, and western blot.
RESULTS AND DISCUSSION: In this study, we identified PARP11 as a host factor that can significantly affect PRV infection. Inhibition of PARP11 and knockout of PARP11 can significantly promoted PRV infection. Subsequently, we further found that PARP11 knockout upregulated the transcription of NXF1 and CRM1, resulting in enhanced transcription of viral genes. Furthermore, we also found that PARP11 knockout could activate the autophagy pathway and suppress the mTOR pathway during PRV infection. These findings could provide insight into the mechanism in which PARP11 participated during PRV infection and offer a potential target to develop anti-PRV therapies.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
Swine
*Herpesvirus 1, Suid/genetics
Cell Line
*Host-Pathogen Interactions
*Gene Knockout Techniques
*Poly(ADP-ribose) Polymerases/metabolism/genetics
CRISPR-Cas Systems
Autophagy
Pseudorabies/virology
Virus Replication
Swine Diseases/virology
TOR Serine-Threonine Kinases/metabolism
RevDate: 2024-10-23
PlasmidScope: a comprehensive plasmid database with rich annotations and online analytical tools.
Nucleic acids research pii:7832352 [Epub ahead of print].
Plasmids are extrachromosomal genetic molecules that replicate independent of chromosomes in bacteria, archaea, and eukaryotic organisms. They contain diverse functional elements and are capable of horizontal gene transfer among hosts. While existing plasmid databases have archived plasmid sequences isolated from individual microorganisms or natural environments, there is a need for a comprehensive, standardized, and annotated plasmid database to address the vast accumulation of plasmid sequences. Here, we propose PlasmidScope (https://plasmid.deepomics.org/), a plasmid database offering comprehensive annotations, automated online analysis, and interactive visualization. PlasmidScope harbors a substantial collection of 852 600 plasmids curated from 10 repositories. Along with consolidated background information, PlasmidScope utilizes 12 state-of-the-art tools and provides comprehensive annotations for the curated plasmids, covering genome completeness, topological structure, mobility, host source, tRNA, tmRNA, signal peptides, transmembrane proteins and CRISPR/Cas systems. PlasmidScope offers diverse functional annotations for its 25 231 059 predicted genes from 9 databases as well as corresponding protein structures predicted by ESMFold. In addition, PlasmidScope integrates online analytical modules and interactive visualization, empowering researchers to delve into the complexities of plasmids.
Additional Links: PMID-39441081
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39441081,
year = {2024},
author = {Li, Y and Feng, X and Chen, X and Yang, S and Zhao, Z and Chen, Y and Li, SC},
title = {PlasmidScope: a comprehensive plasmid database with rich annotations and online analytical tools.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae930},
pmid = {39441081},
issn = {1362-4962},
support = {32061160472//NSFC-RGC Joint Research Scheme/ ; 32300527//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 20220814183301001//Shenzhen Science and Technology Program/ ; },
abstract = {Plasmids are extrachromosomal genetic molecules that replicate independent of chromosomes in bacteria, archaea, and eukaryotic organisms. They contain diverse functional elements and are capable of horizontal gene transfer among hosts. While existing plasmid databases have archived plasmid sequences isolated from individual microorganisms or natural environments, there is a need for a comprehensive, standardized, and annotated plasmid database to address the vast accumulation of plasmid sequences. Here, we propose PlasmidScope (https://plasmid.deepomics.org/), a plasmid database offering comprehensive annotations, automated online analysis, and interactive visualization. PlasmidScope harbors a substantial collection of 852 600 plasmids curated from 10 repositories. Along with consolidated background information, PlasmidScope utilizes 12 state-of-the-art tools and provides comprehensive annotations for the curated plasmids, covering genome completeness, topological structure, mobility, host source, tRNA, tmRNA, signal peptides, transmembrane proteins and CRISPR/Cas systems. PlasmidScope offers diverse functional annotations for its 25 231 059 predicted genes from 9 databases as well as corresponding protein structures predicted by ESMFold. In addition, PlasmidScope integrates online analytical modules and interactive visualization, empowering researchers to delve into the complexities of plasmids.},
}
RevDate: 2024-10-23
CmpDate: 2024-10-23
Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells.
Microbial cell factories, 23(1):289.
BACKGROUND: The high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.
RESULTS: In this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.
CONCLUSIONS: For efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.
Additional Links: PMID-39438921
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39438921,
year = {2024},
author = {Luelf, UJ and Wassing, A and Böhmer, LM and Urlacher, VB},
title = {Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells.},
journal = {Microbial cell factories},
volume = {23},
number = {1},
pages = {289},
pmid = {39438921},
issn = {1475-2859},
support = {031B0362A//Bundesministerium für Bildung und Forschung/ ; 031B0362A//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Lignans/biosynthesis/metabolism ; *Escherichia coli/metabolism/genetics ; *Plasmids/genetics/metabolism ; Furans/metabolism ; Metabolic Engineering/methods ; Biosynthetic Pathways ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.
RESULTS: In this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.
CONCLUSIONS: For efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Lignans/biosynthesis/metabolism
*Escherichia coli/metabolism/genetics
*Plasmids/genetics/metabolism
Furans/metabolism
Metabolic Engineering/methods
Biosynthetic Pathways
CRISPR-Cas Systems
RevDate: 2024-10-24
CmpDate: 2024-10-23
Optimization of a novel expression system for recombinant protein production in CHO cells.
Scientific reports, 14(1):24913.
Chinese hamster ovary (CHO) cells are common mammalian cell lines for expressing recombinant proteins, yet the expression level of recombinant proteins is still hindered. Vector optimization and cell line modification are the key factors to improve the expression of recombinant proteins. In this study, the vector was optimized by adding the regulatory elements Kozak and Leader to the upstream of target gene to detect the transient and stable expression of recombinant proteins. Results indicated that the expression level of target proteins with the addition of regulatory elements was significantly increased compared with the control group. In addition, the inhibition of apoptotic pathway has great potential to increase recombinant protein production, and Apaf1 protein dependent on the mitochondrial apoptosis pathway plays an important role in this respect. The knockout of apoptotic gene Apaf1 in CHO cells can also increase recombinant protein production. Therefore, the vector was optimized by adding regulatory elements, and the cell line was modified by using CRISPR/Cas9 technology to establish a novel CHO cell expression system, which remarkably improved the expression level of recombinant proteins and laid the foundation for the large-scale production of recombinant proteins.
Additional Links: PMID-39438721
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39438721,
year = {2024},
author = {Zhang, J and Du, C and Pan, Y and Zhang, Z and Feng, R and Ma, M and Wang, T},
title = {Optimization of a novel expression system for recombinant protein production in CHO cells.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {24913},
pmid = {39438721},
issn = {2045-2322},
support = {23A310015//the Key Research Projects of Higher Education Institutions of Henan Province/ ; },
mesh = {Animals ; CHO Cells ; *Cricetulus ; *Recombinant Proteins/genetics/metabolism/biosynthesis ; *CRISPR-Cas Systems ; Genetic Vectors/genetics ; Cricetinae ; Apoptosis/genetics ; Gene Expression ; },
abstract = {Chinese hamster ovary (CHO) cells are common mammalian cell lines for expressing recombinant proteins, yet the expression level of recombinant proteins is still hindered. Vector optimization and cell line modification are the key factors to improve the expression of recombinant proteins. In this study, the vector was optimized by adding the regulatory elements Kozak and Leader to the upstream of target gene to detect the transient and stable expression of recombinant proteins. Results indicated that the expression level of target proteins with the addition of regulatory elements was significantly increased compared with the control group. In addition, the inhibition of apoptotic pathway has great potential to increase recombinant protein production, and Apaf1 protein dependent on the mitochondrial apoptosis pathway plays an important role in this respect. The knockout of apoptotic gene Apaf1 in CHO cells can also increase recombinant protein production. Therefore, the vector was optimized by adding regulatory elements, and the cell line was modified by using CRISPR/Cas9 technology to establish a novel CHO cell expression system, which remarkably improved the expression level of recombinant proteins and laid the foundation for the large-scale production of recombinant proteins.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
CHO Cells
*Cricetulus
*Recombinant Proteins/genetics/metabolism/biosynthesis
*CRISPR-Cas Systems
Genetic Vectors/genetics
Cricetinae
Apoptosis/genetics
Gene Expression
RevDate: 2024-10-24
CmpDate: 2024-10-24
CRISPR-Cas12a detection of DNA glycosylases via DNA modification switching.
Chemical communications (Cambridge, England), 60(86):12569-12572.
A programmable CRISPR-Cas12a system for selective detection of various DNA glycosylases is described. By temporarily inactivating Cas12a through the introduction of specific DNA modifications in the complementary DNA strand of Cas12a's crRNA, the system is able to detect the target DNA glycosylases. This approach addresses critical gaps in current CRISPR-Cas12a diagnostics for non-nucleic acid detection beyond the limitations of aptamers.
Additional Links: PMID-39385597
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39385597,
year = {2024},
author = {Li, Y and Yang, X and Dong, Y and Wang, J and Liu, C},
title = {CRISPR-Cas12a detection of DNA glycosylases via DNA modification switching.},
journal = {Chemical communications (Cambridge, England)},
volume = {60},
number = {86},
pages = {12569-12572},
doi = {10.1039/d4cc04180a},
pmid = {39385597},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry ; *DNA Glycosylases/metabolism ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism ; },
abstract = {A programmable CRISPR-Cas12a system for selective detection of various DNA glycosylases is described. By temporarily inactivating Cas12a through the introduction of specific DNA modifications in the complementary DNA strand of Cas12a's crRNA, the system is able to detect the target DNA glycosylases. This approach addresses critical gaps in current CRISPR-Cas12a diagnostics for non-nucleic acid detection beyond the limitations of aptamers.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems/genetics
*DNA/chemistry
*DNA Glycosylases/metabolism
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/metabolism
RevDate: 2024-10-24
CmpDate: 2024-10-24
Development of inducible promoter and CRISPRi plasmids functional in Rickettsia rickettsii.
Journal of bacteriology, 206(10):e0036724.
UNLABELLED: Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.
IMPORTANCE: This work expands upon the genetic toolbox available for R. rickettsii. We describe both an inducible promoter and CRISPRi system compatible with Rickettsia, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma, Orientia, and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms.
Additional Links: PMID-39347571
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39347571,
year = {2024},
author = {Nock, AM and Clark, TR and Hackstadt, T},
title = {Development of inducible promoter and CRISPRi plasmids functional in Rickettsia rickettsii.},
journal = {Journal of bacteriology},
volume = {206},
number = {10},
pages = {e0036724},
doi = {10.1128/jb.00367-24},
pmid = {39347571},
issn = {1098-5530},
support = {AI000977//HHS | NIH | NIAID | Division of Intramural Research (DIR, NIAID)/ ; },
mesh = {*Promoter Regions, Genetic ; *Rickettsia rickettsii/genetics ; *Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; },
abstract = {UNLABELLED: Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.
IMPORTANCE: This work expands upon the genetic toolbox available for R. rickettsii. We describe both an inducible promoter and CRISPRi system compatible with Rickettsia, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma, Orientia, and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Promoter Regions, Genetic
*Rickettsia rickettsii/genetics
*Plasmids/genetics
*Gene Expression Regulation, Bacterial
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
RevDate: 2024-10-24
Gene drive and RNAi technologies: a bio-cultural review of next-generation tools for pest wasp management in New Zealand.
Journal of the Royal Society of New Zealand, 52(5):508-525.
There is a global need for novel, next-generation technologies and techniques to manage pest species. We review work on potential step-changing technologies for large landscape (>1000 hectares) pest management of social Vespula wasps. We also review Māori perspectives on these controls to gauge social and cultural acceptability to research, test and use of novel controls. Approaches discussed are the use of gene silencing (RNAi) and gene drives (CRISPR-Cas 9) involving genetic modification, which has potential for pest control but vary in feasibility, cost, benefits and off-target risks. RNAi may be better suited for wasp control in high-value cropping systems due to scaling inefficiencies. Gene drives offer potential for large-scale control but would require legislative and wide social deliberation due to their status as genetic modification. Both RNAi and gene drives will require consultation with tangata whenua. Māori interest groups agreed that exotic wasps must be controlled and expressed aversion to non-targeted traditional control methods. We present a diversity of opinions in parallel with scientific research underscoring the need for continued dialogue with Māori. Novel biotechnological controls must satisfy a broad range of social and cultural criteria, receive regulatory approval, along with being demonstrated as safe, selective, and cost-effective.
Additional Links: PMID-39440191
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39440191,
year = {2022},
author = {Palmer, S and Dearden, PK and Mercier, OR and King-Hunt, A and Lester, PJ},
title = {Gene drive and RNAi technologies: a bio-cultural review of next-generation tools for pest wasp management in New Zealand.},
journal = {Journal of the Royal Society of New Zealand},
volume = {52},
number = {5},
pages = {508-525},
pmid = {39440191},
issn = {1175-8899},
abstract = {There is a global need for novel, next-generation technologies and techniques to manage pest species. We review work on potential step-changing technologies for large landscape (>1000 hectares) pest management of social Vespula wasps. We also review Māori perspectives on these controls to gauge social and cultural acceptability to research, test and use of novel controls. Approaches discussed are the use of gene silencing (RNAi) and gene drives (CRISPR-Cas 9) involving genetic modification, which has potential for pest control but vary in feasibility, cost, benefits and off-target risks. RNAi may be better suited for wasp control in high-value cropping systems due to scaling inefficiencies. Gene drives offer potential for large-scale control but would require legislative and wide social deliberation due to their status as genetic modification. Both RNAi and gene drives will require consultation with tangata whenua. Māori interest groups agreed that exotic wasps must be controlled and expressed aversion to non-targeted traditional control methods. We present a diversity of opinions in parallel with scientific research underscoring the need for continued dialogue with Māori. Novel biotechnological controls must satisfy a broad range of social and cultural criteria, receive regulatory approval, along with being demonstrated as safe, selective, and cost-effective.},
}
RevDate: 2024-10-22
CRISPR-Cas systems in enterococci.
Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] [Epub ahead of print].
Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37 bp and direct repeat sequences from 25 to 37 bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.
Additional Links: PMID-39438415
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39438415,
year = {2024},
author = {Cabral, AS and Lacerda, FF and Leite, VLM and de Miranda, FM and da Silva, AB and Dos Santos, BA and Lima, JLDC and Teixeira, LM and Neves, FPG},
title = {CRISPR-Cas systems in enterococci.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {},
number = {},
pages = {},
pmid = {39438415},
issn = {1678-4405},
support = {001//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; 308059/2021-2//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; E-26/211.554/2019//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E-26/210.103/2020//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E-26/200.855/2021//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; },
abstract = {Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37 bp and direct repeat sequences from 25 to 37 bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.},
}
RevDate: 2024-10-22
CRISPR/Cas13a-mediated visual detection: A rapid and robust method for early detection of Nosema bombycis in silkworms.
Insect biochemistry and molecular biology pii:S0965-1748(24)00134-6 [Epub ahead of print].
The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.
Additional Links: PMID-39437972
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39437972,
year = {2024},
author = {Wu, YX and Sadiq, S and Jiao, XH and Zhou, XM and Wang, LL and Xie, XR and Khan, I and Wu, P},
title = {CRISPR/Cas13a-mediated visual detection: A rapid and robust method for early detection of Nosema bombycis in silkworms.},
journal = {Insect biochemistry and molecular biology},
volume = {},
number = {},
pages = {104203},
doi = {10.1016/j.ibmb.2024.104203},
pmid = {39437972},
issn = {1879-0240},
abstract = {The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.},
}
RevDate: 2024-10-22
CmpDate: 2024-10-22
[Migration Medicine: infectious and non-infectious diseases].
Deutsche medizinische Wochenschrift (1946), 149(22):1335-1340.
Approximately one third of the German population has a migration background.According to the federal office for migration, in 2022 around 2,7 million people have taken refuge or have immigrated to Germany, causing major challenges for our health system. In this article, important infectious diseases, and non-infectious conditions like hemoglobinopathies are presented. To date, especially the latter are not common in Germany and must therefore move more into focus when taking care of migrants. Furthermore, new treatment options for hemoglobinopathies have been approved in the last couple of years, starting with the introduction of Luspatercept a few years ago for Beta-Thalassemias and in 2023 the introduction of Voxelotor for sickle cell disease. In 2024 the gene therapy with Exagamglogen-Autotemcel using the CRISPR/Cas molecular scissor was authorized as a new promising treatment for both conditions.
Additional Links: PMID-39437825
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39437825,
year = {2024},
author = {Distelmaier, L and Ehrlich, S and Wallrauch, C and von Bergwelt-Baildon, M and Rothe, C},
title = {[Migration Medicine: infectious and non-infectious diseases].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {149},
number = {22},
pages = {1335-1340},
doi = {10.1055/a-2338-7169},
pmid = {39437825},
issn = {1439-4413},
mesh = {Humans ; Germany ; *Communicable Diseases/therapy ; Noncommunicable Diseases/therapy ; },
abstract = {Approximately one third of the German population has a migration background.According to the federal office for migration, in 2022 around 2,7 million people have taken refuge or have immigrated to Germany, causing major challenges for our health system. In this article, important infectious diseases, and non-infectious conditions like hemoglobinopathies are presented. To date, especially the latter are not common in Germany and must therefore move more into focus when taking care of migrants. Furthermore, new treatment options for hemoglobinopathies have been approved in the last couple of years, starting with the introduction of Luspatercept a few years ago for Beta-Thalassemias and in 2023 the introduction of Voxelotor for sickle cell disease. In 2024 the gene therapy with Exagamglogen-Autotemcel using the CRISPR/Cas molecular scissor was authorized as a new promising treatment for both conditions.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
Germany
*Communicable Diseases/therapy
Noncommunicable Diseases/therapy
RevDate: 2024-10-22
CmpDate: 2024-10-22
Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells.
Cell reports methods, 4(10):100882.
The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.
Additional Links: PMID-39437714
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39437714,
year = {2024},
author = {Marsiglia, J and Vaalavirta, K and Knight, E and Nakamura, M and Cong, L and Hughes, NW},
title = {Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells.},
journal = {Cell reports methods},
volume = {4},
number = {10},
pages = {100882},
doi = {10.1016/j.crmeth.2024.100882},
pmid = {39437714},
issn = {2667-2375},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Human/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA Repair ; },
abstract = {The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Genome, Human/genetics
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
DNA Repair
RevDate: 2024-10-23
CmpDate: 2024-10-23
Deployment of tethered gene drive for confined suppression in continuous space requires avoiding drive wave interference.
Molecular ecology, 33(19):e17530.
Gene drives have great potential for suppression of pest populations and removal of exotic invasive species. CRISPR homing suppression drive is a powerful but unconfined drive, posing risks of uncontrolled spread. Thus, developing methods for confining a gene drive is of great significance. Tethered drive combines a confined system such as Toxin-Antidote Recessive Embryo drive with a strong drive such as a homing suppression drive. It can prevent the homing drive from spreading beyond the confined drive and can be constructed readily, giving it good prospects for future development. However, we have found that care must be taken when deploying tethered drive systems in some scenarios. Simulations of tethered drive in a panmictic population model reveal that successful deployment requires a proper release ratio between the two components, tailored to prevent the suppression drive from eliminating the confined system before it has the chance to spread. Spatial models where the population moves over a one-dimensional landscape display a more serious phenomenon of drive wave interference between the two tethered drive components. If the faster suppression drive wave catches up to the confined drive wave, success is still possible, but it is dependent on drive performance and ecological parameters. Two-dimensional simulations further restrict the parameter range for drive success. Thus, careful consideration must be given to drive performance and ecological conditions, as well as specific release proposals for potential application of tethered drive systems.
Additional Links: PMID-39282691
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39282691,
year = {2024},
author = {Feng, R and Champer, J},
title = {Deployment of tethered gene drive for confined suppression in continuous space requires avoiding drive wave interference.},
journal = {Molecular ecology},
volume = {33},
number = {19},
pages = {e17530},
doi = {10.1111/mec.17530},
pmid = {39282691},
issn = {1365-294X},
support = {//Center for Life Sciences/ ; 32270672//National Natural Science Foundation of China/ ; },
mesh = {*Gene Drive Technology/methods ; Animals ; *Introduced Species ; Models, Genetic ; Computer Simulation ; Genetics, Population ; CRISPR-Cas Systems ; },
abstract = {Gene drives have great potential for suppression of pest populations and removal of exotic invasive species. CRISPR homing suppression drive is a powerful but unconfined drive, posing risks of uncontrolled spread. Thus, developing methods for confining a gene drive is of great significance. Tethered drive combines a confined system such as Toxin-Antidote Recessive Embryo drive with a strong drive such as a homing suppression drive. It can prevent the homing drive from spreading beyond the confined drive and can be constructed readily, giving it good prospects for future development. However, we have found that care must be taken when deploying tethered drive systems in some scenarios. Simulations of tethered drive in a panmictic population model reveal that successful deployment requires a proper release ratio between the two components, tailored to prevent the suppression drive from eliminating the confined system before it has the chance to spread. Spatial models where the population moves over a one-dimensional landscape display a more serious phenomenon of drive wave interference between the two tethered drive components. If the faster suppression drive wave catches up to the confined drive wave, success is still possible, but it is dependent on drive performance and ecological parameters. Two-dimensional simulations further restrict the parameter range for drive success. Thus, careful consideration must be given to drive performance and ecological conditions, as well as specific release proposals for potential application of tethered drive systems.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Drive Technology/methods
Animals
*Introduced Species
Models, Genetic
Computer Simulation
Genetics, Population
CRISPR-Cas Systems
RevDate: 2024-10-23
CmpDate: 2024-10-23
DNA self-assembly-boosted transcription amplification coupled with CRISPR/Cas13a system for plant microRNA analysis.
Talanta, 281:126890.
MicroRNAs (miRNAs) play important roles in the growth process of plants, and some food-originated plant miRNAs have potential impacts on human health, which makes the detection of plant miRNAs of great significance. However, plant miRNAs are naturally modified with 2'-O-methyl at the 3'-terminal, which is difficult to be directly quantified by enzyme-catalyzed terminal polymerization protocols. Herein, we have proposed a simple strategy by coupling DNA self-assembly-boosted transcription amplification with CRISPR/Cas13a platform (termed as Cas13a-SATA) for the specific and sensitive detection of plant miRNA. In the Cas13a-SATA, the plant miRNA will mediate DNA self-assembly on the surface of microbeads and then trigger efficient transcription amplification to yield numerous single-stranded RNA (ssRNA) molecules, which can effectively activate the Cas13a trans-cleavage activity to generate intense fluorescence signal in a plant miRNA dosage-responsive manner. Using the Cas13a-SATA, we have realized the sensitive detection of plant miR156a with the limit of detection (LOD) down to 3.8 fM. Furthermore, Cas13a-SATA has been successfully applied to the accurate quantification of miR156a in Arabidopsis and maize, demonstrating its feasibility in analyzing plant miRNAs in real biological samples.
Additional Links: PMID-39277941
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39277941,
year = {2025},
author = {Wang, G and Li, Z},
title = {DNA self-assembly-boosted transcription amplification coupled with CRISPR/Cas13a system for plant microRNA analysis.},
journal = {Talanta},
volume = {281},
number = {},
pages = {126890},
doi = {10.1016/j.talanta.2024.126890},
pmid = {39277941},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Plant/genetics ; DNA/chemistry/genetics ; Transcription, Genetic ; Limit of Detection ; Arabidopsis/genetics ; },
abstract = {MicroRNAs (miRNAs) play important roles in the growth process of plants, and some food-originated plant miRNAs have potential impacts on human health, which makes the detection of plant miRNAs of great significance. However, plant miRNAs are naturally modified with 2'-O-methyl at the 3'-terminal, which is difficult to be directly quantified by enzyme-catalyzed terminal polymerization protocols. Herein, we have proposed a simple strategy by coupling DNA self-assembly-boosted transcription amplification with CRISPR/Cas13a platform (termed as Cas13a-SATA) for the specific and sensitive detection of plant miRNA. In the Cas13a-SATA, the plant miRNA will mediate DNA self-assembly on the surface of microbeads and then trigger efficient transcription amplification to yield numerous single-stranded RNA (ssRNA) molecules, which can effectively activate the Cas13a trans-cleavage activity to generate intense fluorescence signal in a plant miRNA dosage-responsive manner. Using the Cas13a-SATA, we have realized the sensitive detection of plant miR156a with the limit of detection (LOD) down to 3.8 fM. Furthermore, Cas13a-SATA has been successfully applied to the accurate quantification of miR156a in Arabidopsis and maize, demonstrating its feasibility in analyzing plant miRNAs in real biological samples.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*MicroRNAs/genetics
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
RNA, Plant/genetics
DNA/chemistry/genetics
Transcription, Genetic
Limit of Detection
Arabidopsis/genetics
RevDate: 2024-10-23
CmpDate: 2024-10-23
Direct quantification of N[6]-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated CRISPR-Cas12a platform.
Talanta, 281:126806.
As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N[6]-methyladenosine (m[6]A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m[6]A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m[6]A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m[6]A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m[6]A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m[6]A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m[6]A fractions in real biological samples, even in low abundance RNA biomarkers.
Additional Links: PMID-39277937
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39277937,
year = {2025},
author = {Hu, Z and Liu, W and Chen, D and Gao, K and Li, Z},
title = {Direct quantification of N[6]-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated CRISPR-Cas12a platform.},
journal = {Talanta},
volume = {281},
number = {},
pages = {126806},
doi = {10.1016/j.talanta.2024.126806},
pmid = {39277937},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *Adenosine/analogs & derivatives/analysis/chemistry ; *DNA, Catalytic/chemistry/metabolism/genetics ; *RNA/genetics/analysis/chemistry ; Humans ; },
abstract = {As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N[6]-methyladenosine (m[6]A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m[6]A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m[6]A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m[6]A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m[6]A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m[6]A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m[6]A fractions in real biological samples, even in low abundance RNA biomarkers.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems/genetics
*Adenosine/analogs & derivatives/analysis/chemistry
*DNA, Catalytic/chemistry/metabolism/genetics
*RNA/genetics/analysis/chemistry
Humans
RevDate: 2024-10-23
CmpDate: 2024-10-23
Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies.
Talanta, 281:126795.
This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.
Additional Links: PMID-39236519
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39236519,
year = {2025},
author = {Tang, Q and Huang, X and Liu, G and Huang, H and Zhang, J and Wei, J and Gong, Y and Tang, Q and Zhang, K and Liao, X},
title = {Electrochemical detection of MMP-2 with enhanced sensitivity: Utilizing T7 RNA polymerase and CRISPR-Cas12a for neuronal studies.},
journal = {Talanta},
volume = {281},
number = {},
pages = {126795},
doi = {10.1016/j.talanta.2024.126795},
pmid = {39236519},
issn = {1873-3573},
mesh = {Humans ; *Matrix Metalloproteinase 2/genetics/metabolism ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *DNA-Directed RNA Polymerases/metabolism ; *Viral Proteins ; Hep G2 Cells ; Limit of Detection ; },
abstract = {This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Matrix Metalloproteinase 2/genetics/metabolism
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
*DNA-Directed RNA Polymerases/metabolism
*Viral Proteins
Hep G2 Cells
Limit of Detection
RevDate: 2024-10-23
CmpDate: 2024-10-23
CRISPR/dCas13(Rx) Derived RNA N[6]-methyladenosine (m[6]A) Dynamic Modification in Plant.
Advanced science (Weinheim, Baden-Wurttemberg, Germany), 11(39):e2401118.
N[6]-methyladenosine (m[6]A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m[6]A sites of individual transcripts in plants. Here, programmable m[6]A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m[6]A modifications at targeted sites of endo-transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m[6]A levels by 24%-76%, while the TME editor increases m[6]A enrichment, ranging from 1.37- to 2.51-fold. Furthermore, installation and removal of m[6]A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m[6]A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m[6]A editors can be applied to study the function of specific m[6]A modifications and have the potential for future applications in crop improvement.
Additional Links: PMID-39229923
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39229923,
year = {2024},
author = {Yu, L and Alariqi, M and Li, B and Hussain, A and Zhou, H and Wang, Q and Wang, F and Wang, G and Zhu, X and Hui, F and Yang, X and Nie, X and Zhang, X and Jin, S},
title = {CRISPR/dCas13(Rx) Derived RNA N[6]-methyladenosine (m[6]A) Dynamic Modification in Plant.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {39},
pages = {e2401118},
doi = {10.1002/advs.202401118},
pmid = {39229923},
issn = {2198-3844},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 2022ZD0402001-04//Science and Technology Innovation 2030/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; 32272128//National Natural Science Fund of China/ ; },
mesh = {*Adenosine/analogs & derivatives/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Arabidopsis/genetics ; RNA, Plant/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {N[6]-methyladenosine (m[6]A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m[6]A sites of individual transcripts in plants. Here, programmable m[6]A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m[6]A modifications at targeted sites of endo-transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m[6]A levels by 24%-76%, while the TME editor increases m[6]A enrichment, ranging from 1.37- to 2.51-fold. Furthermore, installation and removal of m[6]A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m[6]A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m[6]A editors can be applied to study the function of specific m[6]A modifications and have the potential for future applications in crop improvement.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Adenosine/analogs & derivatives/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Arabidopsis/genetics
RNA, Plant/genetics/metabolism
Plants, Genetically Modified/genetics
RevDate: 2024-10-23
CmpDate: 2024-10-23
RNA-protein interactions reveals the pivotal role of lncRNA1840 in tomato fruit maturation.
The Plant journal : for cell and molecular biology, 120(2):526-539.
Long non-coding RNAs (lncRNAs) play crucial roles in various biological processes in plants. However, the functional mechanism of lncRNAs in fruit ripening, particularly the transition from unripe to ripe stages, remains elusive. One such lncRNA1840, reported by our group, was found to have important role in tomato fruit ripening. In the present study, we gain insight into its functional role in fruit ripening. CRISPR-Cas9 mediated lncRNA1840 mutants caused the delayed tomato fruit ripening. Notably, loss function of lncRNA1840 did not directly impact ethylene signaling but rather delay ethylene synthesis. Transcriptomic analysis revealed differences in the expression of ripening related genes in lncRNA1840 mutants, suggesting that it is involved in gene regulation of fruit ripening. We used Chromatin Isolation by RNA Purification (ChIRP)-Seq to identify lncRNA1840 binding sites on chromatin. ChIRP-seq suggested that lncRNA1840 had occupancy on 40 genes, but none of them is differentially expressed genes in transcriptomic analysis, which indicated lncRNA1840 might indirectly modulate the gene expression. ChIRP-mass spectrometry analysis identified potential protein interactors of lncRNA1840, Pre-mRNA processing splicing factor 8, highlighting its involvement in post-transcriptional regulatory pathways. In summary, lncRNA1840 is key player in tomato plant growth and fruit ripening, with multifaceted roles in gene expression and regulatory networks.
Additional Links: PMID-39226395
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39226395,
year = {2024},
author = {Zhu, G and Li, R and Zhang, L and Ma, L and Li, J and Chen, J and Deng, Z and Yan, S and Li, T and Ren, H and Cui, K and Qu, G and Zhu, B and Fu, D and Luo, Y and Zhu, H},
title = {RNA-protein interactions reveals the pivotal role of lncRNA1840 in tomato fruit maturation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {120},
number = {2},
pages = {526-539},
doi = {10.1111/tpj.16998},
pmid = {39226395},
issn = {1365-313X},
support = {2022YFD2100101//National Key Research and Development Program of China/ ; 1061-00109017//2115 Talent Development Program of China Agricultural University/ ; 32061143022//National Natural Science Foundation of China/ ; 32172639//National Natural Science Foundation of China/ ; 32202558//National Natural Science Foundation of China/ ; 32302623//National Natural Science Foundation of China/ ; },
mesh = {*Solanum lycopersicum/genetics/growth & development/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; *Fruit/genetics/growth & development/metabolism ; *Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Ethylenes/metabolism ; RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; Chromatin/metabolism/genetics ; },
abstract = {Long non-coding RNAs (lncRNAs) play crucial roles in various biological processes in plants. However, the functional mechanism of lncRNAs in fruit ripening, particularly the transition from unripe to ripe stages, remains elusive. One such lncRNA1840, reported by our group, was found to have important role in tomato fruit ripening. In the present study, we gain insight into its functional role in fruit ripening. CRISPR-Cas9 mediated lncRNA1840 mutants caused the delayed tomato fruit ripening. Notably, loss function of lncRNA1840 did not directly impact ethylene signaling but rather delay ethylene synthesis. Transcriptomic analysis revealed differences in the expression of ripening related genes in lncRNA1840 mutants, suggesting that it is involved in gene regulation of fruit ripening. We used Chromatin Isolation by RNA Purification (ChIRP)-Seq to identify lncRNA1840 binding sites on chromatin. ChIRP-seq suggested that lncRNA1840 had occupancy on 40 genes, but none of them is differentially expressed genes in transcriptomic analysis, which indicated lncRNA1840 might indirectly modulate the gene expression. ChIRP-mass spectrometry analysis identified potential protein interactors of lncRNA1840, Pre-mRNA processing splicing factor 8, highlighting its involvement in post-transcriptional regulatory pathways. In summary, lncRNA1840 is key player in tomato plant growth and fruit ripening, with multifaceted roles in gene expression and regulatory networks.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Solanum lycopersicum/genetics/growth & development/metabolism
*RNA, Long Noncoding/genetics/metabolism
*Fruit/genetics/growth & development/metabolism
*Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
Ethylenes/metabolism
RNA, Plant/genetics/metabolism
CRISPR-Cas Systems
Chromatin/metabolism/genetics
RevDate: 2024-10-22
CRISPR-Cas technology in forensic investigations: Principles, applications, and ethical considerations.
Forensic science international. Genetics, 74:103163 pii:S1872-4973(24)00159-5 [Epub ahead of print].
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems are adaptive immune systems originally present in bacteria, where they are essential to protect against external genetic elements, including viruses and plasmids. Taking advantage of this system, CRISPR-Cas-based technologies have emerged as incredible tools for precise genome editing, thus significantly advancing several research fields. Forensic sciences represent a multidisciplinary field that explores scientific methods to investigate and resolve legal issues, particularly criminal investigations and subject identification. Consequently, it plays a critical role in the justice system, providing scientific evidence to support judicial investigations. Although less explored, CRISPR-Cas-based methodologies demonstrate strong potential in the field of forensic sciences due to their high accuracy and sensitivity, including DNA profiling and identification, interpretation of crime scene investigations, detection of food contamination or fraud, and other aspects related to environmental forensics. However, using CRISPR-Cas-based methodologies in human samples raises several ethical issues and concerns regarding the potential misuse of individual genetic information. In this manuscript, we provide an overview of potential applications of CRISPR-Cas-based methodologies in several areas of forensic sciences and discuss the legal implications that challenge their routine implementation in this research field.
Additional Links: PMID-39437497
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39437497,
year = {2024},
author = {Sobral, AF and Dinis-Oliveira, RJ and Barbosa, DJ},
title = {CRISPR-Cas technology in forensic investigations: Principles, applications, and ethical considerations.},
journal = {Forensic science international. Genetics},
volume = {74},
number = {},
pages = {103163},
doi = {10.1016/j.fsigen.2024.103163},
pmid = {39437497},
issn = {1878-0326},
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems are adaptive immune systems originally present in bacteria, where they are essential to protect against external genetic elements, including viruses and plasmids. Taking advantage of this system, CRISPR-Cas-based technologies have emerged as incredible tools for precise genome editing, thus significantly advancing several research fields. Forensic sciences represent a multidisciplinary field that explores scientific methods to investigate and resolve legal issues, particularly criminal investigations and subject identification. Consequently, it plays a critical role in the justice system, providing scientific evidence to support judicial investigations. Although less explored, CRISPR-Cas-based methodologies demonstrate strong potential in the field of forensic sciences due to their high accuracy and sensitivity, including DNA profiling and identification, interpretation of crime scene investigations, detection of food contamination or fraud, and other aspects related to environmental forensics. However, using CRISPR-Cas-based methodologies in human samples raises several ethical issues and concerns regarding the potential misuse of individual genetic information. In this manuscript, we provide an overview of potential applications of CRISPR-Cas-based methodologies in several areas of forensic sciences and discuss the legal implications that challenge their routine implementation in this research field.},
}
RevDate: 2024-10-22
CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.
The CRISPR journal [Epub ahead of print].
Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.
Additional Links: PMID-39436817
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39436817,
year = {2024},
author = {Cotter, CJ and Trinh, CT},
title = {CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0052},
pmid = {39436817},
issn = {2573-1602},
abstract = {Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.},
}
RevDate: 2024-10-22
CmpDate: 2024-10-22
Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.
The CRISPR journal, 7(5):258-271.
Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34[+] cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.
Additional Links: PMID-39436283
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39436283,
year = {2024},
author = {Ritter, MU and Nasri, M and Dannenmann, B and Mir, P and Secker, B and Amend, D and Klimiankou, M and Welte, K and Skokowa, J},
title = {Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {258-271},
doi = {10.1089/crispr.2024.0006},
pmid = {39436283},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; *Neutropenia/genetics/therapy/congenital ; *Congenital Bone Marrow Failure Syndromes/genetics/therapy ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Mutation ; Alleles ; Induced Pluripotent Stem Cells/metabolism ; Recombinational DNA Repair/genetics ; Leukocyte Elastase ; },
abstract = {Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34[+] cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
Humans
*Neutropenia/genetics/therapy/congenital
*Congenital Bone Marrow Failure Syndromes/genetics/therapy
*CRISPR-Cas Systems
*Genetic Therapy/methods
*Mutation
Alleles
Induced Pluripotent Stem Cells/metabolism
Recombinational DNA Repair/genetics
Leukocyte Elastase
RevDate: 2024-10-22
CmpDate: 2024-10-22
Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.
The CRISPR journal, 7(5):293-304.
Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.
Additional Links: PMID-39436282
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39436282,
year = {2024},
author = {Niwa, R and Matsumoto, T and Liu, AY and Kawato, M and Kondo, T and Tsukita, K and Gee, P and Inoue, H and Maurissen, TL and Woltjen, K},
title = {Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {293-304},
doi = {10.1089/crispr.2024.0028},
pmid = {39436282},
issn = {2573-1602},
mesh = {*Induced Pluripotent Stem Cells/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Alleles ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Apolipoproteins E/genetics ; Alzheimer Disease/genetics/therapy ; },
abstract = {Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Induced Pluripotent Stem Cells/metabolism
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Alleles
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Apolipoproteins E/genetics
Alzheimer Disease/genetics/therapy
RevDate: 2024-10-22
CmpDate: 2024-10-22
Five Years of Progress in CRISPR Clinical Trials (2019-2024).
The CRISPR journal, 7(5):227-230.
In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both in vivo and ex vivo gene and cell therapies.
Additional Links: PMID-39436279
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39436279,
year = {2024},
author = {Davies, K and Philippidis, A and Barrangou, R},
title = {Five Years of Progress in CRISPR Clinical Trials (2019-2024).},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {227-230},
doi = {10.1089/crispr.2024.0081},
pmid = {39436279},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Clinical Trials as Topic ; *Genetic Therapy/methods ; Anemia, Sickle Cell/therapy/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell- and Tissue-Based Therapy/methods ; },
abstract = {In July 2019, Victoria Gray became the first patient with sickle cell disease to receive a CRISPR-based cell therapy as a volunteer in the exa-cel clinical trial, sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Barely four years later, the ensuing therapy, branded as Casgevy, received approval from regulatory agencies in Europe, the United States, and the Middle East, ushering in a new era of CRISPR-based medicines. During this period, scores of other clinical trials have been launched, including many actively recruiting patients across phase 1, phase 2, and phase 3 clinical trials around the world. In this brief Perspective, we collate the latest information on therapeutic clinical trials featuring CRISPR, base and prime editing, across a range of both in vivo and ex vivo gene and cell therapies.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Clinical Trials as Topic
*Genetic Therapy/methods
Anemia, Sickle Cell/therapy/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Cell- and Tissue-Based Therapy/methods
RevDate: 2024-10-22
CmpDate: 2024-10-22
Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.
The CRISPR journal, 7(5):283-292.
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.
Additional Links: PMID-39436278
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39436278,
year = {2024},
author = {Li, VR and Wu, T and Tadych, A and Wong, A and Zhang, Z},
title = {Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {283-292},
doi = {10.1089/crispr.2024.0020},
pmid = {39436278},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Variation ; Genome, Human ; Clustered Regularly Interspaced Short Palindromic Repeats ; Prealbumin/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Amyloidosis/genetics/therapy ; Amyloid Neuropathies, Familial ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Variation
Genome, Human
Clustered Regularly Interspaced Short Palindromic Repeats
Prealbumin/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Amyloidosis/genetics/therapy
Amyloid Neuropathies, Familial
RevDate: 2024-10-22
CRISPR-edited, cell-based future-proof meat and seafood to enhance global food security and nutrition.
Cytotechnology, 76(6):619-652.
Food security is a major concern due to the growing population and climate change. A method for increasing food production is the use of modern biotechnology, such as cell culture, marker-assisted selection, and genetic engineering. Cellular agriculture has enabled the production of cell-cultivated meat in bioreactors that mimic the properties of conventional meat. Furthermore, 3D food printing technology has improved food production by adding new nutritional and organoleptic properties. Marker-assisted selection and genetic engineering could play an important role in producing animals and crops with desirable traits. Therefore, integrating cellular agriculture with genetic engineering technology could be a potential strategy for the production of cell-based meat and seafood with high health benefits in the future. This review highlights the production of cell-cultivated meat derived from a variety of species, including livestock, birds, fish, and marine crustaceans. It also investigates the application of genetic engineering methods, such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), in the context of cellular agriculture. Moreover, it examines aspects such as food safety, regulatory considerations, and consumer acceptance of genetically engineered cell-cultivated meat and seafood.
Additional Links: PMID-39435422
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39435422,
year = {2024},
author = {Chandrababu, A and Puthumana, J},
title = {CRISPR-edited, cell-based future-proof meat and seafood to enhance global food security and nutrition.},
journal = {Cytotechnology},
volume = {76},
number = {6},
pages = {619-652},
pmid = {39435422},
issn = {0920-9069},
abstract = {Food security is a major concern due to the growing population and climate change. A method for increasing food production is the use of modern biotechnology, such as cell culture, marker-assisted selection, and genetic engineering. Cellular agriculture has enabled the production of cell-cultivated meat in bioreactors that mimic the properties of conventional meat. Furthermore, 3D food printing technology has improved food production by adding new nutritional and organoleptic properties. Marker-assisted selection and genetic engineering could play an important role in producing animals and crops with desirable traits. Therefore, integrating cellular agriculture with genetic engineering technology could be a potential strategy for the production of cell-based meat and seafood with high health benefits in the future. This review highlights the production of cell-cultivated meat derived from a variety of species, including livestock, birds, fish, and marine crustaceans. It also investigates the application of genetic engineering methods, such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), in the context of cellular agriculture. Moreover, it examines aspects such as food safety, regulatory considerations, and consumer acceptance of genetically engineered cell-cultivated meat and seafood.},
}
RevDate: 2024-10-22
Data on the analysis of draft genome sequence of Raoultella ornithinolytica isolate carrying antimicrobial resistance genes, plasmid and CRISPR-Cas system.
Data in brief, 57:110973.
Environmental bacterial species Raoultella ornithinolytica is an emerging pathogen becoming increasingly important in causing human infections. Thus far, the clinical isolates of this species have not exhibited multidrug resistance very often, but some reports underline the necessity for continuous monitoring of this potentially dangerous pathogen. Currently, epidemiological surveillance and antimicrobial resistance investigations of any bacterial pathogen usually rely on whole genome sequencing, which is becoming more affordable while providing increasingly important data in the recent years. However, R. ornithinolytica genomic information is scantily presented in public databases. Here, we report, to the best of our knowledge, the first whole genome sequence and corresponding raw data for a clinical R. ornithinolytica isolate from Russian Federation, which carried antimicrobial resistance (AMR) genes, virulence factors, one plasmid, and CRISPR-Cas system of type I-F. The data provided will facilitate epidemiological surveillance and antimicrobial resistance monitoring of this emerging pathogen.
Additional Links: PMID-39435365
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39435365,
year = {2024},
author = {Karpenko, A and Mikhaylova, Y and Shelenkov, A and Tutelyan, A and Akimkin, V},
title = {Data on the analysis of draft genome sequence of Raoultella ornithinolytica isolate carrying antimicrobial resistance genes, plasmid and CRISPR-Cas system.},
journal = {Data in brief},
volume = {57},
number = {},
pages = {110973},
pmid = {39435365},
issn = {2352-3409},
abstract = {Environmental bacterial species Raoultella ornithinolytica is an emerging pathogen becoming increasingly important in causing human infections. Thus far, the clinical isolates of this species have not exhibited multidrug resistance very often, but some reports underline the necessity for continuous monitoring of this potentially dangerous pathogen. Currently, epidemiological surveillance and antimicrobial resistance investigations of any bacterial pathogen usually rely on whole genome sequencing, which is becoming more affordable while providing increasingly important data in the recent years. However, R. ornithinolytica genomic information is scantily presented in public databases. Here, we report, to the best of our knowledge, the first whole genome sequence and corresponding raw data for a clinical R. ornithinolytica isolate from Russian Federation, which carried antimicrobial resistance (AMR) genes, virulence factors, one plasmid, and CRISPR-Cas system of type I-F. The data provided will facilitate epidemiological surveillance and antimicrobial resistance monitoring of this emerging pathogen.},
}
RevDate: 2024-10-22
CmpDate: 2024-10-22
The RNA m[5]C methyltransferase NSUN1 modulates human malaria gene expression during intraerythrocytic development.
Frontiers in cellular and infection microbiology, 14:1474229.
INTRODUCTION: Plasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m[5]C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein.
METHODS: An efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments.
RESULTS: The efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly down-regulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor.
DISCUSSION: PfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum. It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor.
Additional Links: PMID-39435184
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39435184,
year = {2024},
author = {Tang, R and Fan, Y and Lu, B and Jiang, Q and Cheng, X and Zhang, Z and Shen, L and Shang, X},
title = {The RNA m[5]C methyltransferase NSUN1 modulates human malaria gene expression during intraerythrocytic development.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1474229},
pmid = {39435184},
issn = {2235-2988},
mesh = {*Plasmodium falciparum/genetics/growth & development ; Humans ; *Erythrocytes/parasitology ; *Protozoan Proteins/genetics/metabolism ; *Malaria, Falciparum/parasitology ; Methyltransferases/genetics/metabolism ; Gene Expression Regulation ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Gene Expression Profiling ; Transcriptome ; Gene Editing ; Methylation ; },
abstract = {INTRODUCTION: Plasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m[5]C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein.
METHODS: An efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments.
RESULTS: The efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly down-regulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor.
DISCUSSION: PfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum. It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Plasmodium falciparum/genetics/growth & development
Humans
*Erythrocytes/parasitology
*Protozoan Proteins/genetics/metabolism
*Malaria, Falciparum/parasitology
Methyltransferases/genetics/metabolism
Gene Expression Regulation
CRISPR-Cas Systems
Gene Knockdown Techniques
Gene Expression Profiling
Transcriptome
Gene Editing
Methylation
RevDate: 2024-10-22
CRISPR/Cas and Argonaute-powered lateral flow assay for pathogens detection.
Critical reviews in food science and nutrition [Epub ahead of print].
Pathogens contamination is a pressing global public issue that has garnered significant attention worldwide, especially in light of recent outbreaks of foodborne illnesses. Programmable nucleases like CRISPR/Cas and Argonaute hold promise as tools for nucleic acid testing owning to programmability and the precise target sequence specificity, which has been utilized for the development pathogens detection. At present, fluorescence, as the main signal output method, provides a simple response mode for sensing analysis. However, the dependence of fluorescence output on large instruments and correct analysis of output data limited its use in remote areas. Lateral flow strips (LFS), emerging as a novel flexible substrate, offer a plethora of advantages, encompassing easy-to-use, rapidity, visualization, low-cost, portability, etc. The integration of CRISPR/Cas and Argonaute with LFS, lateral flow assay (LFA), rendered a new and on-site mode for pathogens detection. In the review, we introduced two programmable nucleases CRISPR/Cas and Argonaute, followed by the structure, principle and advantages of LFA. Then diversified engineering detection pattens for viruses, bacteria, parasites, and fungi based on CRISPR/Cas and Argonaute were introduced and summarized. Finally, the challenge and perspectives involved in on-site diagnostic assays were discussed.
Additional Links: PMID-39434421
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39434421,
year = {2024},
author = {Li, Y and Zhao, L and Ma, L and Bai, Y and Feng, F},
title = {CRISPR/Cas and Argonaute-powered lateral flow assay for pathogens detection.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-23},
doi = {10.1080/10408398.2024.2416473},
pmid = {39434421},
issn = {1549-7852},
abstract = {Pathogens contamination is a pressing global public issue that has garnered significant attention worldwide, especially in light of recent outbreaks of foodborne illnesses. Programmable nucleases like CRISPR/Cas and Argonaute hold promise as tools for nucleic acid testing owning to programmability and the precise target sequence specificity, which has been utilized for the development pathogens detection. At present, fluorescence, as the main signal output method, provides a simple response mode for sensing analysis. However, the dependence of fluorescence output on large instruments and correct analysis of output data limited its use in remote areas. Lateral flow strips (LFS), emerging as a novel flexible substrate, offer a plethora of advantages, encompassing easy-to-use, rapidity, visualization, low-cost, portability, etc. The integration of CRISPR/Cas and Argonaute with LFS, lateral flow assay (LFA), rendered a new and on-site mode for pathogens detection. In the review, we introduced two programmable nucleases CRISPR/Cas and Argonaute, followed by the structure, principle and advantages of LFA. Then diversified engineering detection pattens for viruses, bacteria, parasites, and fungi based on CRISPR/Cas and Argonaute were introduced and summarized. Finally, the challenge and perspectives involved in on-site diagnostic assays were discussed.},
}
RevDate: 2024-10-21
CmpDate: 2024-10-22
Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae.
BMC genomics, 25(1):985.
BACKGROUND: Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.
RESULTS: In this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens.
CONCLUSION: We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.
Additional Links: PMID-39433986
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39433986,
year = {2024},
author = {Debatisse, K and Niault, T and Peeters, S and Maire, A and Toktas, B and Darracq, B and Baharoglu, Z and Bikard, D and Mazel, D and Loot, C},
title = {Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {985},
pmid = {39433986},
issn = {1471-2164},
support = {ANR-21-CE12-0002-01//ANR/ ; ANR-19CE12-0001//ANR/ ; ANR-21-CE12-0002-01//ANR/ ; EQU202103012569//FRM/ ; },
mesh = {*Vibrio cholerae/genetics ; *CRISPR-Cas Systems ; Cholera/microbiology/epidemiology ; Genome, Bacterial ; Pandemics ; Humans ; High-Throughput Nucleotide Sequencing ; Gene Library ; },
abstract = {BACKGROUND: Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.
RESULTS: In this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens.
CONCLUSION: We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Vibrio cholerae/genetics
*CRISPR-Cas Systems
Cholera/microbiology/epidemiology
Genome, Bacterial
Pandemics
Humans
High-Throughput Nucleotide Sequencing
Gene Library
RevDate: 2024-10-21
Multiplex Genomic Tagging of Mammalian ATG8s to Study Autophagy.
The Journal of biological chemistry pii:S0021-9258(24)02410-4 [Epub ahead of print].
Atg8 proteins play a crucial role in autophagy. There is a single Atg8 isoform in yeast, while mammals have up to seven homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16, implicated in various stages along the pathway. However, the intricacies among GABARAP proteins are complex and require a more precise delineation. Here, we introduce a new cellular platform to study autophagy using CRISPR/Cas9-mediated tagging of endogenous genes of the GABARAP subfamily with different fluorescent proteins. This platform allows robust examination of autophagy by flow cytometry of cell populations and monitoring of GABARAP homologs at single-cell resolution using fluorescence microscopy. Strikingly, the simultaneous labeling of the different endogenous GABARAPs allows the identification and isolation of autophagosomes differentially marked by these proteins. Using this system, we found that the different GABARAPs are associated with different autophagosomes. We argue that this new cellular platform will be crucial in studying the unique roles of individual GABARAP proteins in autophagy and other putative cellular processes.
Additional Links: PMID-39433127
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39433127,
year = {2024},
author = {Goldin-Azulay, K and Fraiberg, M and Trofimyuk, O and Levin, Y and Reuven, N and Kopitman, E and Elazar, Z},
title = {Multiplex Genomic Tagging of Mammalian ATG8s to Study Autophagy.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {107908},
doi = {10.1016/j.jbc.2024.107908},
pmid = {39433127},
issn = {1083-351X},
abstract = {Atg8 proteins play a crucial role in autophagy. There is a single Atg8 isoform in yeast, while mammals have up to seven homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16, implicated in various stages along the pathway. However, the intricacies among GABARAP proteins are complex and require a more precise delineation. Here, we introduce a new cellular platform to study autophagy using CRISPR/Cas9-mediated tagging of endogenous genes of the GABARAP subfamily with different fluorescent proteins. This platform allows robust examination of autophagy by flow cytometry of cell populations and monitoring of GABARAP homologs at single-cell resolution using fluorescence microscopy. Strikingly, the simultaneous labeling of the different endogenous GABARAPs allows the identification and isolation of autophagosomes differentially marked by these proteins. Using this system, we found that the different GABARAPs are associated with different autophagosomes. We argue that this new cellular platform will be crucial in studying the unique roles of individual GABARAP proteins in autophagy and other putative cellular processes.},
}
RevDate: 2024-10-19
ENZYMATIC TOOLS FOR MITOCHONDRIAL GENOME MANIPULATION.
Biochimie pii:S0300-9084(24)00239-6 [Epub ahead of print].
Mutations in mitochondrial DNA (mtDNA) can manifest phenotypically as a wide range of neuromuscular and neurodegenerative pathologies that are currently only managed symptomatically without addressing the root cause. A promising approach is the development of molecular tools aimed at mtDNA cutting or editing. Unlike nuclear DNA, a cell can have hundreds or even thousands of mitochondrial genomes, and mutations can be present either in all of them or only in a subset. Consequently, the developed tools are aimed at reducing the number of copies of mutant mtDNA or editing mutant nucleotides. Despite some progress in the field of mitochondrial genome editing in human cells, working with model animals is still limited due to the complexity of their creation. Furthermore, not all existing editing systems can be easily adapted to function within mitochondria. In this review, we evaluate the mtDNA editing tools available today, with a particular focus on specific mtDNA mutations linked to hereditary mitochondrial diseases, aiming to provide an in-depth understanding of both the opportunities and hurdles to the development of mitochondrial genome editing technologies.
Additional Links: PMID-39426703
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39426703,
year = {2024},
author = {Rimskaya, B and Shebanov, N and Entelis, N and Mazunin, I},
title = {ENZYMATIC TOOLS FOR MITOCHONDRIAL GENOME MANIPULATION.},
journal = {Biochimie},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.biochi.2024.10.013},
pmid = {39426703},
issn = {1638-6183},
abstract = {Mutations in mitochondrial DNA (mtDNA) can manifest phenotypically as a wide range of neuromuscular and neurodegenerative pathologies that are currently only managed symptomatically without addressing the root cause. A promising approach is the development of molecular tools aimed at mtDNA cutting or editing. Unlike nuclear DNA, a cell can have hundreds or even thousands of mitochondrial genomes, and mutations can be present either in all of them or only in a subset. Consequently, the developed tools are aimed at reducing the number of copies of mutant mtDNA or editing mutant nucleotides. Despite some progress in the field of mitochondrial genome editing in human cells, working with model animals is still limited due to the complexity of their creation. Furthermore, not all existing editing systems can be easily adapted to function within mitochondria. In this review, we evaluate the mtDNA editing tools available today, with a particular focus on specific mtDNA mutations linked to hereditary mitochondrial diseases, aiming to provide an in-depth understanding of both the opportunities and hurdles to the development of mitochondrial genome editing technologies.},
}
RevDate: 2024-10-18
CmpDate: 2024-10-18
Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology.
Methods in molecular biology (Clifton, N.J.), 2865:241-257.
Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, select cells with a specified phenotype, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human B-cell lymphoma cell lines but are highly amenable for other experimental purposes.
Additional Links: PMID-39424727
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39424727,
year = {2025},
author = {Bolomsky, A and Choi, J and Phelan, JD},
title = {Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2865},
number = {},
pages = {241-257},
pmid = {39424727},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; *Phenotype ; Cell Line, Tumor ; Lymphoma/genetics ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Lentivirus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genomics/methods ; },
abstract = {Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, select cells with a specified phenotype, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human B-cell lymphoma cell lines but are highly amenable for other experimental purposes.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
*Phenotype
Cell Line, Tumor
Lymphoma/genetics
Genotype
High-Throughput Nucleotide Sequencing/methods
Lentivirus/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genomics/methods
RevDate: 2024-10-18
Detection of the jellyfish Chrysaora pacifica by RPA-CRISPR-Cas12a environmental DNA (eDNA) assay and its evaluation through field validation and comparative eDNA analyses.
The Science of the total environment pii:S0048-9697(24)07102-X [Epub ahead of print].
Climate-driven environmental changes and anthropogenic activities can result in the proliferation of non-indigenous aquatic species such as jellyfish that may cause envenomation and various ecological disruptions. Here we developed a two-step RPA-CRISPR-Cas12a eDNA assay, consisting of target eDNA amplification followed by a CRISPR-Cas12 reaction, for the early detection of Chrysaora pacifica, a jellyfish species often considered non-indigenous to South Korea. The assay demonstrated high sensitivity, with a detection limit of two copies COI/μL for eDNA derived from C. pacifica, using target specific RPA primers and crRNA sequences. Field validation of the assay using eDNA samples from Jinhae Bay collected over eight months of time-series monitoring, revealed temporal distribution of the jellyfish which correlated with results of digital polymerase chain reaction (dPCR) and eDNA metabarcoding. The C. pacifica eDNA assays were also corroborated (R-square 0.7891) by reports from a citizen science-based jellyfish-monitoring program operated by the National Institute of Fisheries Science, South Korea. Our RPA-CRISPR-Cas eDNA assay can therefore, be an efficient alternative to traditional tools for the early detection of outbreaks of non-indigenous or harmful species in marine ecosystems.
Additional Links: PMID-39423898
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39423898,
year = {2024},
author = {Kim, K and Maji, UJ and Shim, KY and Yeo, IC and Jeong, CB},
title = {Detection of the jellyfish Chrysaora pacifica by RPA-CRISPR-Cas12a environmental DNA (eDNA) assay and its evaluation through field validation and comparative eDNA analyses.},
journal = {The Science of the total environment},
volume = {},
number = {},
pages = {176945},
doi = {10.1016/j.scitotenv.2024.176945},
pmid = {39423898},
issn = {1879-1026},
abstract = {Climate-driven environmental changes and anthropogenic activities can result in the proliferation of non-indigenous aquatic species such as jellyfish that may cause envenomation and various ecological disruptions. Here we developed a two-step RPA-CRISPR-Cas12a eDNA assay, consisting of target eDNA amplification followed by a CRISPR-Cas12 reaction, for the early detection of Chrysaora pacifica, a jellyfish species often considered non-indigenous to South Korea. The assay demonstrated high sensitivity, with a detection limit of two copies COI/μL for eDNA derived from C. pacifica, using target specific RPA primers and crRNA sequences. Field validation of the assay using eDNA samples from Jinhae Bay collected over eight months of time-series monitoring, revealed temporal distribution of the jellyfish which correlated with results of digital polymerase chain reaction (dPCR) and eDNA metabarcoding. The C. pacifica eDNA assays were also corroborated (R-square 0.7891) by reports from a citizen science-based jellyfish-monitoring program operated by the National Institute of Fisheries Science, South Korea. Our RPA-CRISPR-Cas eDNA assay can therefore, be an efficient alternative to traditional tools for the early detection of outbreaks of non-indigenous or harmful species in marine ecosystems.},
}
RevDate: 2024-10-22
CmpDate: 2024-10-22
Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.
The CRISPR journal, 7(5):272-282.
Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.
Additional Links: PMID-39387256
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39387256,
year = {2024},
author = {Xinyue, Z and Li, S and Yujie, W and Yingcai, D and Changhao, B and Xueli, Z},
title = {Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {272-282},
doi = {10.1089/crispr.2024.0016},
pmid = {39387256},
issn = {2573-1602},
mesh = {Humans ; HEK293 Cells ; *Lentivirus/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
HEK293 Cells
*Lentivirus/genetics
*CRISPR-Cas Systems
*Genetic Vectors/genetics
*Gene Knockout Techniques/methods
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats
RevDate: 2024-10-22
CmpDate: 2024-10-22
Affordable Pricing of CRISPR Treatments is a Pressing Ethical Imperative.
The CRISPR journal, 7(5):220-226.
Casgevy, the world's first approved CRISPR-based cell therapy, has been priced at $2.2 million per patient. Although this hefty price tag was widely anticipated, the extremely high cost of this and other cell and gene therapies poses a major ethical issue in terms of equitable access and global health. In this Perspective, we argue that lowering the prices of future CRISPR therapies is an urgent ethical imperative. Although we focus on Casgevy as a case study, much of our analysis can be extrapolated to the controversies over affordable access to other gene and cell therapies. First, we explain why this first-of-its-kind CRISPR therapy might be so expensive. We then analyze the ethical issues of equity and global health of early CRISPR treatments. Next, we discuss potential solutions to lower the prices of CRISPR gene therapies. We conclude that the approval of CRISPR transforms our obligations of justice and compels us to bring future gene therapies to the maximum possible number of patients with serious genetic diseases at affordable prices.
Additional Links: PMID-39392045
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39392045,
year = {2024},
author = {Rueda, J and de Miguel Beriain, Í and Montoliu, L},
title = {Affordable Pricing of CRISPR Treatments is a Pressing Ethical Imperative.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {220-226},
doi = {10.1089/crispr.2024.0042},
pmid = {39392045},
issn = {2573-1602},
mesh = {Humans ; *Genetic Therapy/ethics/economics ; *CRISPR-Cas Systems ; *Gene Editing/ethics/economics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell- and Tissue-Based Therapy/ethics/economics ; Costs and Cost Analysis ; },
abstract = {Casgevy, the world's first approved CRISPR-based cell therapy, has been priced at $2.2 million per patient. Although this hefty price tag was widely anticipated, the extremely high cost of this and other cell and gene therapies poses a major ethical issue in terms of equitable access and global health. In this Perspective, we argue that lowering the prices of future CRISPR therapies is an urgent ethical imperative. Although we focus on Casgevy as a case study, much of our analysis can be extrapolated to the controversies over affordable access to other gene and cell therapies. First, we explain why this first-of-its-kind CRISPR therapy might be so expensive. We then analyze the ethical issues of equity and global health of early CRISPR treatments. Next, we discuss potential solutions to lower the prices of CRISPR gene therapies. We conclude that the approval of CRISPR transforms our obligations of justice and compels us to bring future gene therapies to the maximum possible number of patients with serious genetic diseases at affordable prices.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Genetic Therapy/ethics/economics
*CRISPR-Cas Systems
*Gene Editing/ethics/economics
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell- and Tissue-Based Therapy/ethics/economics
Costs and Cost Analysis
RevDate: 2024-10-22
CmpDate: 2024-10-22
Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.
PLoS genetics, 20(10):e1011438 pii:PGENETICS-D-24-00299.
Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.
Additional Links: PMID-39388480
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39388480,
year = {2024},
author = {Chen, X and Perry, S and Fan, Z and Wang, B and Loxterkamp, E and Wang, S and Hu, J and Dickman, D and Han, C},
title = {Tissue-specific knockout in the Drosophila neuromuscular system reveals ESCRT's role in formation of synapse-derived extracellular vesicles.},
journal = {PLoS genetics},
volume = {20},
number = {10},
pages = {e1011438},
doi = {10.1371/journal.pgen.1011438},
pmid = {39388480},
issn = {1553-7404},
mesh = {Animals ; *Neuromuscular Junction/metabolism/genetics ; *Endosomal Sorting Complexes Required for Transport/genetics/metabolism ; *Extracellular Vesicles/metabolism/genetics ; *CRISPR-Cas Systems ; *Drosophila Proteins/genetics/metabolism ; *Motor Neurons/metabolism ; Drosophila melanogaster/genetics ; Gene Knockout Techniques ; SNARE Proteins/metabolism/genetics ; Synapses/metabolism/genetics ; Drosophila/genetics ; },
abstract = {Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Neuromuscular Junction/metabolism/genetics
*Endosomal Sorting Complexes Required for Transport/genetics/metabolism
*Extracellular Vesicles/metabolism/genetics
*CRISPR-Cas Systems
*Drosophila Proteins/genetics/metabolism
*Motor Neurons/metabolism
Drosophila melanogaster/genetics
Gene Knockout Techniques
SNARE Proteins/metabolism/genetics
Synapses/metabolism/genetics
Drosophila/genetics
RevDate: 2024-10-22
CmpDate: 2024-10-22
Genome Editing Therapy for the Blood: Ex Vivo Success and In Vivo Prospects.
The CRISPR journal, 7(5):231-248.
Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via in vivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to in vivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.
Additional Links: PMID-39324895
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39324895,
year = {2024},
author = {George, CA and Sahu, SU and de Oñate, L and Souza, BSF and Wilson, RC},
title = {Genome Editing Therapy for the Blood: Ex Vivo Success and In Vivo Prospects.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {231-248},
doi = {10.1089/crispr.2024.0036},
pmid = {39324895},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism ; *CRISPR-Cas Systems ; Hematopoietic Stem Cell Transplantation/methods ; Animals ; T-Lymphocytes ; },
abstract = {Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient's HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via in vivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to in vivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
Humans
*Genetic Therapy/methods
*Hematopoietic Stem Cells/metabolism
*CRISPR-Cas Systems
Hematopoietic Stem Cell Transplantation/methods
Animals
T-Lymphocytes
RevDate: 2024-10-22
CmpDate: 2024-10-22
Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.
The CRISPR journal, 7(5):249-257.
Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.
Additional Links: PMID-39324883
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39324883,
year = {2024},
author = {Mohan, R and Haga, SB},
title = {Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.},
journal = {The CRISPR journal},
volume = {7},
number = {5},
pages = {249-257},
doi = {10.1089/crispr.2024.0011},
pmid = {39324883},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; *Clinical Trials as Topic ; *CRISPR-Cas Systems ; United States ; },
abstract = {Genome editing technologies have become widely used research tools. To assess the rate of growth with respect to federal funding of gene editing projects, we analyzed publicly available data retrieved from the NIH RePORTER and Clinicaltrials.gov databases. We identified 6,111 awards between 1977 and 2023, the majority being extramural, investigator-driven R (noneducational) awards (66.7%). There was an average growth rate of 40% between 2008 and 2022, and the biggest increase in awards was observed between 2017 and 2018 (doubling from 140 to 280). Five administering institutes/centers accounted for more than 60% of awards with the highest number of awards from the National Cancer Institute (20.0%). The majority of clinical trials involving some type of genome editing (75%) started in or after 2020. This analysis illuminates the rapid and widespread growth of gene editing research across disciplines and the eventual launch of clinical trials using gene editing tools.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
Humans
*Clinical Trials as Topic
*CRISPR-Cas Systems
United States
RevDate: 2024-10-22
CmpDate: 2024-10-22
Investigation of physiological roles of UDP-glycosyltransferase UGT76F2 in auxin homeostasis through the TAA-YUCCA auxin biosynthesis pathway.
Bioscience, biotechnology, and biochemistry, 88(11):1326-1335.
Cellular auxin (indole-3-acetic acid, IAA) levels are coordinately regulated by IAA biosynthesis and inactivation. IAA is synthesized through sequential reactions by two enzymes, TAA1 and YUCCA, in a linear indole-3-pyruvic acid (IPA) pathway. TAA1 converts tryptophan to IPA, and YUCCA catalyzes the oxidative decarboxylation of IPA into IAA. Arabidopsis UDP-glycosyltransferase UGT76F2 (At3g55710) was previously reported to catalyze the glycosylation of IPA and consequently modulate IAA levels. We carefully analyzed the physiological roles of UGT76F2 and its close homolog UGT76F1 (At3g55700) in IAA homeostasis. We generated two independent ugt76f1 ugt76f2 double null Arabidopsis mutants (ugt76f1f2) with a 2.7 kb deletion, along with two independent ugt76f2 single null mutants by CRISPR/Cas9 gene editing technology. Surprisingly, these null mutants exhibited indistinguishable phenotypes from the wild-type seedlings under our laboratory conditions. Our results indicate that UGT76F1 and UGT76F2 do not play important roles in regulating IAA biosynthesis via IPA glycosylation.
Additional Links: PMID-39232210
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39232210,
year = {2024},
author = {Harada, M and Kubotsu, T and Agui, T and Dai, X and Zhao, Y and Kasahara, H and Hayashi, KI},
title = {Investigation of physiological roles of UDP-glycosyltransferase UGT76F2 in auxin homeostasis through the TAA-YUCCA auxin biosynthesis pathway.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {88},
number = {11},
pages = {1326-1335},
doi = {10.1093/bbb/zbae124},
pmid = {39232210},
issn = {1347-6947},
support = {JP23H02151//Japan Society for the Promotion of Science/ ; },
mesh = {*Indoleacetic Acids/metabolism ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Homeostasis ; *Glycosyltransferases/metabolism/genetics ; Glycosylation ; Gene Expression Regulation, Plant ; Mutation ; CRISPR-Cas Systems ; Phenotype ; Indoles/metabolism ; },
abstract = {Cellular auxin (indole-3-acetic acid, IAA) levels are coordinately regulated by IAA biosynthesis and inactivation. IAA is synthesized through sequential reactions by two enzymes, TAA1 and YUCCA, in a linear indole-3-pyruvic acid (IPA) pathway. TAA1 converts tryptophan to IPA, and YUCCA catalyzes the oxidative decarboxylation of IPA into IAA. Arabidopsis UDP-glycosyltransferase UGT76F2 (At3g55710) was previously reported to catalyze the glycosylation of IPA and consequently modulate IAA levels. We carefully analyzed the physiological roles of UGT76F2 and its close homolog UGT76F1 (At3g55700) in IAA homeostasis. We generated two independent ugt76f1 ugt76f2 double null Arabidopsis mutants (ugt76f1f2) with a 2.7 kb deletion, along with two independent ugt76f2 single null mutants by CRISPR/Cas9 gene editing technology. Surprisingly, these null mutants exhibited indistinguishable phenotypes from the wild-type seedlings under our laboratory conditions. Our results indicate that UGT76F1 and UGT76F2 do not play important roles in regulating IAA biosynthesis via IPA glycosylation.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Indoleacetic Acids/metabolism
*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/genetics/metabolism
*Homeostasis
*Glycosyltransferases/metabolism/genetics
Glycosylation
Gene Expression Regulation, Plant
Mutation
CRISPR-Cas Systems
Phenotype
Indoles/metabolism
RevDate: 2024-10-22
CmpDate: 2024-10-22
Rice transcription factor DPF regulates stress-induced biosynthesis of diterpenoid phytoalexins.
Bioscience, biotechnology, and biochemistry, 88(11):1344-1348.
Diterpenoid Phytoalexin Factor (DPF) is a key transcription factor involved in diterpenoid phytoalexin (DP) biosynthesis under non-stressed conditions in rice (Oryza sativa L.). Using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9, DPF knockout rice lines were generated. Treatments with abiotic stresses (copper chloride, ultraviolet light, and jasmonic acid) and biotic stress (blast fungus infection) to the knockout lines revealed that the DPF positively regulates stress-induced DP biosynthesis.
Additional Links: PMID-39227176
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39227176,
year = {2024},
author = {Ishikawa, K and Yamamura, C and Miyamoto, K and Kanda, Y and Inoue, H and Okada, K and Kamakura, T and Mori, M},
title = {Rice transcription factor DPF regulates stress-induced biosynthesis of diterpenoid phytoalexins.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {88},
number = {11},
pages = {1344-1348},
doi = {10.1093/bbb/zbae118},
pmid = {39227176},
issn = {1347-6947},
support = {JP17K07782//Japan Society for the Promotion of Science/ ; },
mesh = {*Phytoalexins ; *Oryza/metabolism/microbiology/genetics ; *Sesquiterpenes/metabolism ; *Transcription Factors/genetics/metabolism ; *Stress, Physiological ; *Plant Proteins/genetics/metabolism ; *Diterpenes/metabolism ; Gene Expression Regulation, Plant ; Oxylipins/metabolism ; Cyclopentanes/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Ultraviolet Rays ; },
abstract = {Diterpenoid Phytoalexin Factor (DPF) is a key transcription factor involved in diterpenoid phytoalexin (DP) biosynthesis under non-stressed conditions in rice (Oryza sativa L.). Using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9, DPF knockout rice lines were generated. Treatments with abiotic stresses (copper chloride, ultraviolet light, and jasmonic acid) and biotic stress (blast fungus infection) to the knockout lines revealed that the DPF positively regulates stress-induced DP biosynthesis.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Phytoalexins
*Oryza/metabolism/microbiology/genetics
*Sesquiterpenes/metabolism
*Transcription Factors/genetics/metabolism
*Stress, Physiological
*Plant Proteins/genetics/metabolism
*Diterpenes/metabolism
Gene Expression Regulation, Plant
Oxylipins/metabolism
Cyclopentanes/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems
Ultraviolet Rays
RevDate: 2024-10-22
CmpDate: 2024-10-22
Knockout of OsWOX13 moderately delays flowering in rice under natural long-day conditions.
Bioscience, biotechnology, and biochemistry, 88(11):1307-1315.
Plants are sensitive to photoperiods and are also equipped with systems to adjust their flowering time in response to various changes in the environment and developmental hormones. In the present study, previously generated rice OsWOX13 overexpression and newly generated OsWOX13 knockout lines constructed via CRISPR/Cas9 technology flowered 10 days earlier and 4-6 days later than the wild type, respectively. Quantitative real-time polymerase chain reaction analyses revealed that OsWOX13 might be involved in drought escape responses through the b-ZIP TRANSCRIPTION FACTOR 23 signaling pathway during rice flowering via photoperiod signaling genes such as Grain number, plant height and heading date 7, Early heading date 1, RICE FLOWERING LOCUS T1, Heading date 3a, and MADS14. Future investigations of OsWOX13 may provide insight into how plants adjust their flowering under stress conditions and how OsWOX13 could be precisely controlled to achieve maximum productivity in rice breeding.
Additional Links: PMID-39164217
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39164217,
year = {2024},
author = {Kim, YK},
title = {Knockout of OsWOX13 moderately delays flowering in rice under natural long-day conditions.},
journal = {Bioscience, biotechnology, and biochemistry},
volume = {88},
number = {11},
pages = {1307-1315},
doi = {10.1093/bbb/zbae115},
pmid = {39164217},
issn = {1347-6947},
support = {RS-2023-00241813//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics/growth & development/physiology ; *Flowers/genetics/growth & development ; *Photoperiod ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Droughts ; },
abstract = {Plants are sensitive to photoperiods and are also equipped with systems to adjust their flowering time in response to various changes in the environment and developmental hormones. In the present study, previously generated rice OsWOX13 overexpression and newly generated OsWOX13 knockout lines constructed via CRISPR/Cas9 technology flowered 10 days earlier and 4-6 days later than the wild type, respectively. Quantitative real-time polymerase chain reaction analyses revealed that OsWOX13 might be involved in drought escape responses through the b-ZIP TRANSCRIPTION FACTOR 23 signaling pathway during rice flowering via photoperiod signaling genes such as Grain number, plant height and heading date 7, Early heading date 1, RICE FLOWERING LOCUS T1, Heading date 3a, and MADS14. Future investigations of OsWOX13 may provide insight into how plants adjust their flowering under stress conditions and how OsWOX13 could be precisely controlled to achieve maximum productivity in rice breeding.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Oryza/genetics/growth & development/physiology
*Flowers/genetics/growth & development
*Photoperiod
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
*Gene Knockout Techniques
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
Droughts
RevDate: 2024-10-22
CmpDate: 2024-10-22
PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.
Plant biotechnology journal, 22(11):3164-3174.
Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.
Additional Links: PMID-39031643
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39031643,
year = {2024},
author = {Zheng, Z and Liu, T and Chai, N and Zeng, D and Zhang, R and Wu, Y and Hang, J and Liu, Y and Deng, Q and Tan, J and Liu, J and Xie, X and Liu, YG and Zhu, Q},
title = {PhieDBEs: a DBD-containing, PAM-flexible, high-efficiency dual base editor toolbox with wide targeting scope for use in plants.},
journal = {Plant biotechnology journal},
volume = {22},
number = {11},
pages = {3164-3174},
doi = {10.1111/pbi.14438},
pmid = {39031643},
issn = {1467-7652},
support = {pdjh2023a0077//Special Funds for the Cultivation of Guangdong College Students' Scientific and Technological Innovation "Climbing Program Special Funds"/ ; 2019B030302006//Major Program of Guangdong Basic and Applied Research/ ; 31921004//National Natural Science Foundation of China/ ; 31991222//National Natural Science Foundation of China/ ; 32272120//National Natural Science Foundation of China/ ; 2022YFF1002802//National Key Research and Development Program of China/ ; NT2021002//Laboratory of Lingnan Modern Agriculture Project/ ; 2022SDZG05//Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province/ ; },
mesh = {*Gene Editing/methods ; *Oryza/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Gene Editing/methods
*Oryza/genetics/metabolism
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Adenosine Deaminase/genetics/metabolism
RevDate: 2024-10-22
CmpDate: 2024-10-22
Trans-complementation of the viral movement protein mediates efficient expression of large target genes via a tobacco mosaic virus vector.
Plant biotechnology journal, 22(11):2957-2970.
The development of plant virus-based expression systems has expanded rapidly owing to their potential applications in gene functional and disease resistance research, and industrial production of pharmaceutical proteins. However, the low yield of certain proteins, especially high-molecular-mass proteins, restricts the production scale. In this study, we observed that the tobacco mosaic virus (TMV)-mediated expression of a foreign protein was correlated with the amount of the movement protein (MP) and developed a TMV-derived pAT-[trans]MP vector system incorporating trans-complementation expression of MP. The system is capable of efficient expression of exogenous proteins, in particular those with a high molecular mass, and enables simultaneous expression of two target molecules. Furthermore, viral expression of competent CRISPR-Cas9 protein and construction of CRISPR-Cas9-mediated gene-editing system in a single pAT-[trans]MP construct was achieved. The results demonstrated a novel role for TMV-MP in enhancing the accumulation of a foreign protein produced from the viral vector or a binary expression system. Further investigation of the mechanism underlying this role will be beneficial for optimization of plant viral vectors with broad applications.
Additional Links: PMID-38923265
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38923265,
year = {2024},
author = {Huang, W and Zhang, Y and Xiao, N and Zhao, W and Shi, Y and Fang, R},
title = {Trans-complementation of the viral movement protein mediates efficient expression of large target genes via a tobacco mosaic virus vector.},
journal = {Plant biotechnology journal},
volume = {22},
number = {11},
pages = {2957-2970},
doi = {10.1111/pbi.14418},
pmid = {38923265},
issn = {1467-7652},
support = {31972238//National Natural Science Foundation of China/ ; //Ministry of Agriculture and Rural Affairs of China/ ; },
mesh = {*Tobacco Mosaic Virus/genetics ; *Genetic Vectors/genetics ; *Plant Viral Movement Proteins/genetics/metabolism ; *Nicotiana/genetics/virology/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The development of plant virus-based expression systems has expanded rapidly owing to their potential applications in gene functional and disease resistance research, and industrial production of pharmaceutical proteins. However, the low yield of certain proteins, especially high-molecular-mass proteins, restricts the production scale. In this study, we observed that the tobacco mosaic virus (TMV)-mediated expression of a foreign protein was correlated with the amount of the movement protein (MP) and developed a TMV-derived pAT-[trans]MP vector system incorporating trans-complementation expression of MP. The system is capable of efficient expression of exogenous proteins, in particular those with a high molecular mass, and enables simultaneous expression of two target molecules. Furthermore, viral expression of competent CRISPR-Cas9 protein and construction of CRISPR-Cas9-mediated gene-editing system in a single pAT-[trans]MP construct was achieved. The results demonstrated a novel role for TMV-MP in enhancing the accumulation of a foreign protein produced from the viral vector or a binary expression system. Further investigation of the mechanism underlying this role will be beneficial for optimization of plant viral vectors with broad applications.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Tobacco Mosaic Virus/genetics
*Genetic Vectors/genetics
*Plant Viral Movement Proteins/genetics/metabolism
*Nicotiana/genetics/virology/metabolism
CRISPR-Cas Systems/genetics
Gene Editing/methods
Plants, Genetically Modified/genetics
RevDate: 2024-10-21
CmpDate: 2024-10-18
A neurotransmitter atlas of C. elegans males and hermaphrodites.
eLife, 13:.
Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.
Additional Links: PMID-39422452
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39422452,
year = {2024},
author = {Wang, C and Vidal, B and Sural, S and Loer, C and Aguilar, GR and Merritt, DM and Toker, IA and Vogt, MC and Cros, CC and Hobert, O},
title = {A neurotransmitter atlas of C. elegans males and hermaphrodites.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39422452},
issn = {2050-084X},
support = {P40 OD010440/OD/NIH HHS/United States ; R37 NS039996/NS/NINDS NIH HHS/United States ; Office of Research Infrastructure Programs P40 OD010440/NH/NIH HHS/United States ; NS039996/NH/NIH HHS/United States ; R01 NS039996/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Neurotransmitter Agents/metabolism ; Male ; Neurons/metabolism ; CRISPR-Cas Systems ; },
abstract = {Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the Caenorhabditis elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells, most notably in gonadal cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel sites of monoaminergic neurotransmitter uptake. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Caenorhabditis elegans/genetics/metabolism
*Neurotransmitter Agents/metabolism
Male
Neurons/metabolism
CRISPR-Cas Systems
RevDate: 2024-10-21
CmpDate: 2024-10-21
Visual fluorescence detection of Listeria monocytogenes with CRISPR-Cas12a aptasensor.
Analytical and bioanalytical chemistry, 416(26):5779-5789.
Listeria monocytogenes (L. monocytogenes) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes. In the absence of L. monocytogenes, the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a's trans-cleavage capability. Conversely, in the presence of L. monocytogenes, the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes, with a lowest detection limit (LOD) of 57.15 CFU/mL and a linear range of 4×10[2] to 4×10[7] CFU/mL (R[2]=0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples.
Additional Links: PMID-39212695
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39212695,
year = {2024},
author = {Zhang, R and Wang, Y and Wang, X and Ren, H and Du, J and Yang, Y and Hu, X and Shi, R and Zhang, B and Li, C and Lu, S and Li, Y and Liu, Z and Hu, P},
title = {Visual fluorescence detection of Listeria monocytogenes with CRISPR-Cas12a aptasensor.},
journal = {Analytical and bioanalytical chemistry},
volume = {416},
number = {26},
pages = {5779-5789},
pmid = {39212695},
issn = {1618-2650},
support = {No. 2023YFD1801000//National Key Research and Development Program of China/ ; KLVEB-KFKT-08//State Key Laboratory for Animal Disease Control and Prevention/ ; },
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; *Aptamers, Nucleotide/chemistry ; *Limit of Detection ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Food Microbiology/methods ; Bacterial Proteins/genetics/chemistry ; Fluorescence ; CRISPR-Associated Proteins/genetics/chemistry ; Endodeoxyribonucleases/chemistry ; Spectrometry, Fluorescence/methods ; },
abstract = {Listeria monocytogenes (L. monocytogenes) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes. In the absence of L. monocytogenes, the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a's trans-cleavage capability. Conversely, in the presence of L. monocytogenes, the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes, with a lowest detection limit (LOD) of 57.15 CFU/mL and a linear range of 4×10[2] to 4×10[7] CFU/mL (R[2]=0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Listeria monocytogenes/isolation & purification/genetics
*Aptamers, Nucleotide/chemistry
*Limit of Detection
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Food Microbiology/methods
Bacterial Proteins/genetics/chemistry
Fluorescence
CRISPR-Associated Proteins/genetics/chemistry
Endodeoxyribonucleases/chemistry
Spectrometry, Fluorescence/methods
RevDate: 2024-10-21
CmpDate: 2024-10-21
A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development.
Science China. Life sciences, 67(11):2297-2306.
The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
Additional Links: PMID-39110403
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39110403,
year = {2024},
author = {Zhang, L and Cao, SM and Wu, H and Yan, M and Li, J and Chen, LL},
title = {A CRISPR/RfxCas13d-mediated strategy for efficient RNA knockdown in mouse embryonic development.},
journal = {Science China. Life sciences},
volume = {67},
number = {11},
pages = {2297-2306},
pmid = {39110403},
issn = {1869-1889},
mesh = {Animals ; *Embryonic Development/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Messenger/genetics/metabolism ; RNA, Long Noncoding/genetics/metabolism ; RNA/genetics ; Female ; RNA, Circular/genetics ; Gene Expression Regulation, Developmental ; Male ; Embryo, Mammalian/metabolism ; },
abstract = {The growing variety of RNA classes, such as mRNAs, lncRNAs, and circRNAs, plays pivotal roles in both developmental processes and various pathophysiological conditions. Nonetheless, our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels. In this study, we combined the CRISPR-RfxCas13d system with sperm-like stem cell-mediated semi-cloning techniques, which enabled the suppressed expression of different RNA species. This approach was employed to interfere with the expression of three types of RNA molecules: Sfmbt2 mRNA, Fendrr lncRNA, and circMan1a2(2,3,4,5,6). The results confirmed the critical roles of these RNAs in embryonic development, as their loss led to observable phenotypes, including embryonic lethality, delayed embryonic development, and embryo resorption. In summary, our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Embryonic Development/genetics
Mice
*CRISPR-Cas Systems/genetics
*Gene Knockdown Techniques/methods
RNA, Messenger/genetics/metabolism
RNA, Long Noncoding/genetics/metabolism
RNA/genetics
Female
RNA, Circular/genetics
Gene Expression Regulation, Developmental
Male
Embryo, Mammalian/metabolism
RevDate: 2024-10-21
CmpDate: 2024-10-21
Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.
Journal of chemical ecology, 50(9-10):573-580.
Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.
Additional Links: PMID-38913104
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38913104,
year = {2024},
author = {Maruoka, T and Shirai, Y and Daimon, T and Fujii, R and Dannoura, M and Seidl-Adams, I and Mori, N and Yoshinaga, N},
title = {Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.},
journal = {Journal of chemical ecology},
volume = {50},
number = {9-10},
pages = {573-580},
pmid = {38913104},
issn = {1573-1561},
support = {JPMJSP2110//JST SPRING/ ; 17H05029, 26850063, 23880014//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Spodoptera/genetics ; *Larva/genetics/metabolism/growth & development ; *Nitrogen/metabolism ; Insect Proteins/metabolism/genetics ; Glutamine/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Fatty Acids/metabolism ; Hydrolysis ; },
abstract = {Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Spodoptera/genetics
*Larva/genetics/metabolism/growth & development
*Nitrogen/metabolism
Insect Proteins/metabolism/genetics
Glutamine/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems
Fatty Acids/metabolism
Hydrolysis
RevDate: 2024-10-21
CmpDate: 2024-10-21
CRISPR/Cas9 Gene Editing: A Novel Approach Towards Alzheimer's Disease Treatment.
CNS & neurological disorders drug targets, 23(12):1405-1424.
In defiance of the vast amount of information regarding Alzheimer's disease (AD) that has been learned over the past thirty years, progress toward developing an effective therapy has been difficult. A neurological ailment that progresses and cannot be reversed is Alzheimer's disease, which shows neurofibrillary tangles, beta-amyloid plaque, and a lack of cognitive processes that is created by tau protein clumps with hyperphosphorylation that finally advances to neuronal damage without a recognized treatment, which has stimulated research into new therapeutic strategies. The protein CAS9 is linked to CRISPR, which is a clustered Regularly Interspaced Short Palindromic Repeat that inactivates or corrects a gene by recognizing a gene sequence that produces a doublestranded break has enchanted a whole amount of interest towards its potency to cure gene sequences in AD. The novel CRISPR-Cas9 applications for developing in vitro and in vivo models to the benefit of AD investigation and therapies are thoroughly analyzed in this work. The discussion will also touch on the creation of delivery methods, which is a significant obstacle to the therapeutic use of CRISPR/Cas9 technology. By concentrating on specific genes, such as those that are significant early- onset AD risk factors and late-onset AD risk factors, like the apolipoprotein E4 (APOE4) gene, this study aims to evaluate the potential application of CRISPR/Cas9 as a possible treatment for AD.
Additional Links: PMID-38716549
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38716549,
year = {2024},
author = {Tripathi, S and Sharma, Y and Rane, R and Kumar, D},
title = {CRISPR/Cas9 Gene Editing: A Novel Approach Towards Alzheimer's Disease Treatment.},
journal = {CNS & neurological disorders drug targets},
volume = {23},
number = {12},
pages = {1405-1424},
pmid = {38716549},
issn = {1996-3181},
mesh = {*Alzheimer Disease/therapy/genetics ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Genetic Therapy/methods ; },
abstract = {In defiance of the vast amount of information regarding Alzheimer's disease (AD) that has been learned over the past thirty years, progress toward developing an effective therapy has been difficult. A neurological ailment that progresses and cannot be reversed is Alzheimer's disease, which shows neurofibrillary tangles, beta-amyloid plaque, and a lack of cognitive processes that is created by tau protein clumps with hyperphosphorylation that finally advances to neuronal damage without a recognized treatment, which has stimulated research into new therapeutic strategies. The protein CAS9 is linked to CRISPR, which is a clustered Regularly Interspaced Short Palindromic Repeat that inactivates or corrects a gene by recognizing a gene sequence that produces a doublestranded break has enchanted a whole amount of interest towards its potency to cure gene sequences in AD. The novel CRISPR-Cas9 applications for developing in vitro and in vivo models to the benefit of AD investigation and therapies are thoroughly analyzed in this work. The discussion will also touch on the creation of delivery methods, which is a significant obstacle to the therapeutic use of CRISPR/Cas9 technology. By concentrating on specific genes, such as those that are significant early- onset AD risk factors and late-onset AD risk factors, like the apolipoprotein E4 (APOE4) gene, this study aims to evaluate the potential application of CRISPR/Cas9 as a possible treatment for AD.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Alzheimer Disease/therapy/genetics
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Genetic Therapy/methods
RevDate: 2024-10-18
CmpDate: 2024-10-18
Genome-wide CRISPR screening identifies the pivotal role of ANKRD42 in colorectal cancer metastasis through EMT regulation.
IUBMB life, 76(10):803-819.
Colorectal cancer (CRC), a pervasive and lethal malignancy of gastrointestinal cancer, imposes significant challenges due to the occurrence of distant metastasis in advanced stages. Understanding the intricate regulatory mechanisms driving CRC distant metastasis is of paramount importance. CRISPR-Cas9 screening has emerged as a powerful tool for investigating tumor initiation and progression. However, its application in studying CRC distant metastasis remains largely unexplored. To establish a model that faithfully recapitulates CRC liver metastasis in patients, we developed an in vivo genome-wide CRISPR-Cas9 screening approach using a spleen-injected liver metastasis mouse model. Through comprehensive screening of a whole-genome sgRNA library, we identified ANKRD42 as a pivotal regulatory gene facilitating CRC liver metastasis. Analysis of the TCGA database and our clinical cohorts unveiled heightened ANKRD42 expression in metastases. At the cellular level, the attenuation of ANKRD42 impaired the migration and invasion processes of tumor cells. In vivo experiments further validated these observations, highlighting the diminished liver metastatic capacity of tumor cells upon ANKRD42 knockdown. To unravel the specific mechanisms by which ANKRD42 regulates CRC distant metastasis, we leveraged patient-derived organoid (PDO) models. Depleting ANKRD42 in PDOs sourced from liver metastases precipitated the downregulation of pivotal genes linked to epithelial-mesenchymal transition (EMT), including CDH2 and SNAI2, thereby effectively suppressing tumor metastasis. This study not only establishes a conceptual framework but also identifies potential therapeutic avenues for advanced-stage distant metastasis in CRC patients.
Additional Links: PMID-38822625
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid38822625,
year = {2024},
author = {Liu, S and Zhang, Z and Wang, Z and Li, J and Shen, L},
title = {Genome-wide CRISPR screening identifies the pivotal role of ANKRD42 in colorectal cancer metastasis through EMT regulation.},
journal = {IUBMB life},
volume = {76},
number = {10},
pages = {803-819},
doi = {10.1002/iub.2855},
pmid = {38822625},
issn = {1521-6551},
support = {2022YFC2505006//National Key Research and Development Program of China/ ; A002239//Science Foundation of Peking University Cancer Hospital/ ; BJCH2024GG03//Science Foundation of Peking University Cancer Hospital/ ; 2021M700288//Postdoctoral Science Foundation of China/ ; 2023SGGZ071//Scientific Research Fund for the Demonstration Project of Public Hospital Reform and Quality Development (Gastrointestinal Tumor) that is approved by Peking University Cancer Hospital (Inner Mongolia Campus)/ ; },
mesh = {*Colorectal Neoplasms/pathology/genetics ; *Epithelial-Mesenchymal Transition/genetics ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Neoplastic ; *Liver Neoplasms/secondary/genetics/pathology/metabolism ; Cell Movement ; Neoplasm Metastasis ; Cell Proliferation ; Cell Line, Tumor ; },
abstract = {Colorectal cancer (CRC), a pervasive and lethal malignancy of gastrointestinal cancer, imposes significant challenges due to the occurrence of distant metastasis in advanced stages. Understanding the intricate regulatory mechanisms driving CRC distant metastasis is of paramount importance. CRISPR-Cas9 screening has emerged as a powerful tool for investigating tumor initiation and progression. However, its application in studying CRC distant metastasis remains largely unexplored. To establish a model that faithfully recapitulates CRC liver metastasis in patients, we developed an in vivo genome-wide CRISPR-Cas9 screening approach using a spleen-injected liver metastasis mouse model. Through comprehensive screening of a whole-genome sgRNA library, we identified ANKRD42 as a pivotal regulatory gene facilitating CRC liver metastasis. Analysis of the TCGA database and our clinical cohorts unveiled heightened ANKRD42 expression in metastases. At the cellular level, the attenuation of ANKRD42 impaired the migration and invasion processes of tumor cells. In vivo experiments further validated these observations, highlighting the diminished liver metastatic capacity of tumor cells upon ANKRD42 knockdown. To unravel the specific mechanisms by which ANKRD42 regulates CRC distant metastasis, we leveraged patient-derived organoid (PDO) models. Depleting ANKRD42 in PDOs sourced from liver metastases precipitated the downregulation of pivotal genes linked to epithelial-mesenchymal transition (EMT), including CDH2 and SNAI2, thereby effectively suppressing tumor metastasis. This study not only establishes a conceptual framework but also identifies potential therapeutic avenues for advanced-stage distant metastasis in CRC patients.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Colorectal Neoplasms/pathology/genetics
*Epithelial-Mesenchymal Transition/genetics
Animals
Humans
Mice
*CRISPR-Cas Systems
*Gene Expression Regulation, Neoplastic
*Liver Neoplasms/secondary/genetics/pathology/metabolism
Cell Movement
Neoplasm Metastasis
Cell Proliferation
Cell Line, Tumor
RevDate: 2024-10-19
Context effects on repair of 5'-overhang DNA double-strand breaks induced by Cas12a in Arabidopsis.
Plant direct, 8(10):e70009.
Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination, and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5' overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5'-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end-joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single-strand annealing recombination.
Additional Links: PMID-39421463
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39421463,
year = {2024},
author = {Lageix, S and Hernandez, M and Gallego, ME and Verbeke, J and Bidet, Y and Viala, S and White, CI},
title = {Context effects on repair of 5'-overhang DNA double-strand breaks induced by Cas12a in Arabidopsis.},
journal = {Plant direct},
volume = {8},
number = {10},
pages = {e70009},
pmid = {39421463},
issn = {2475-4455},
abstract = {Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination, and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5' overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5'-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end-joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single-strand annealing recombination.},
}
RevDate: 2024-10-18
Fusing Allosteric Ribozymes with CRISPR-Cas12a for Efficient Diagnostics of Small Molecule Targets.
Small methods [Epub ahead of print].
The CRISPR-Cas systems are adopted as powerful molecular tools for not only genetic manipulation but also point-of-care diagnostics. However, methods to enable diagnostics of non-nucleic-acid targets with these systems are still limited. Herein, by fusing ligand-dependent allosteric ribozymes with CRISPR-Cas12a, a derived CRISPR-Cas system is created for efficient quantitative analysis of non-nucleic-acid targets in 1-2 h. On two different small molecules, the system's generality, reliability and accuracy is demonstrated, and show that the well operability of this system can enable high-throughput detection of a small molecule in blood samples. The system can be further converted to rely on allosteric deoxyribozyme instead of allosteric ribozyme to recognize non-nucleic-acid targets and transduce the signal to CRISPR-Cas12a for amplification, likely making it easier for storage and more consistent in data generation as DNA possess a stability advantage over RNA. This (deoxy)ribozyme-assisted CRISPR-Cas12a system anticipates that it can facilitate bioanalysis in various scientific and clinical settings and further drive the development of clinical translation.
Additional Links: PMID-39420829
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39420829,
year = {2024},
author = {Guo, L and Zhang, S and Du, X and Zhou, M and Gu, H},
title = {Fusing Allosteric Ribozymes with CRISPR-Cas12a for Efficient Diagnostics of Small Molecule Targets.},
journal = {Small methods},
volume = {},
number = {},
pages = {e2401236},
doi = {10.1002/smtd.202401236},
pmid = {39420829},
issn = {2366-9608},
support = {2020YFA0908901//National Key Research and Development Program of China/ ; 22XD1421500//Program of Shanghai Academic Research Leader/ ; 82121002//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR-Cas systems are adopted as powerful molecular tools for not only genetic manipulation but also point-of-care diagnostics. However, methods to enable diagnostics of non-nucleic-acid targets with these systems are still limited. Herein, by fusing ligand-dependent allosteric ribozymes with CRISPR-Cas12a, a derived CRISPR-Cas system is created for efficient quantitative analysis of non-nucleic-acid targets in 1-2 h. On two different small molecules, the system's generality, reliability and accuracy is demonstrated, and show that the well operability of this system can enable high-throughput detection of a small molecule in blood samples. The system can be further converted to rely on allosteric deoxyribozyme instead of allosteric ribozyme to recognize non-nucleic-acid targets and transduce the signal to CRISPR-Cas12a for amplification, likely making it easier for storage and more consistent in data generation as DNA possess a stability advantage over RNA. This (deoxy)ribozyme-assisted CRISPR-Cas12a system anticipates that it can facilitate bioanalysis in various scientific and clinical settings and further drive the development of clinical translation.},
}
RevDate: 2024-10-18
CmpDate: 2024-10-18
Targeting mRNA-coding genes in prostate cancer using CRISPR/Cas9 technology with a special focus on androgen receptor signaling.
Cell communication and signaling : CCS, 22(1):504.
BACKGROUND: Prostate cancer is among prevalent cancers in men. Numerous strategies have been proposed to intervene with the important prostate cancer-related signaling pathways. Among the most promising strategies is CRISPR/Cas9 strategy. This strategy has been used to modify expression of a number of genes in prostate cancer cells.
AIMS: This review summarizes the most recent progresses in the application of CRISPR/Cas9 strategy in modification of prostate cancer-related phenotypes with an especial focus on pathways related to androgen receptor signaling.
CONCLUSION: CRISPR/Cas9 technology has successfully targeted several genes in the prostate cancer cells. Moreover, the efficiency of this technique in reducing tumor burden has been tested in animal models of prostate cancer. Most of targeted genes have been related with the androgen receptor signaling. Targeted modulation of these genes have affected growth of castration-resistant prostate cancer. PI3K/AKT/mTOR signaling and immune response-related genes have been other targets that have been successfully modulated by CRISPR/Cas9 technology in prostate cancer. Based on the rapid translation of this technology into the clinical application, it is anticipated that novel treatments based on this technique change the outcome of this malignancy in future.
Additional Links: PMID-39420406
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39420406,
year = {2024},
author = {Tabibian, M and Moghaddam, FS and Motevaseli, E and Ghafouri-Fard, S},
title = {Targeting mRNA-coding genes in prostate cancer using CRISPR/Cas9 technology with a special focus on androgen receptor signaling.},
journal = {Cell communication and signaling : CCS},
volume = {22},
number = {1},
pages = {504},
pmid = {39420406},
issn = {1478-811X},
mesh = {Male ; Humans ; *CRISPR-Cas Systems/genetics ; *Receptors, Androgen/genetics/metabolism ; *Prostatic Neoplasms/genetics/metabolism/pathology ; *Signal Transduction/genetics ; Animals ; RNA, Messenger/genetics/metabolism ; },
abstract = {BACKGROUND: Prostate cancer is among prevalent cancers in men. Numerous strategies have been proposed to intervene with the important prostate cancer-related signaling pathways. Among the most promising strategies is CRISPR/Cas9 strategy. This strategy has been used to modify expression of a number of genes in prostate cancer cells.
AIMS: This review summarizes the most recent progresses in the application of CRISPR/Cas9 strategy in modification of prostate cancer-related phenotypes with an especial focus on pathways related to androgen receptor signaling.
CONCLUSION: CRISPR/Cas9 technology has successfully targeted several genes in the prostate cancer cells. Moreover, the efficiency of this technique in reducing tumor burden has been tested in animal models of prostate cancer. Most of targeted genes have been related with the androgen receptor signaling. Targeted modulation of these genes have affected growth of castration-resistant prostate cancer. PI3K/AKT/mTOR signaling and immune response-related genes have been other targets that have been successfully modulated by CRISPR/Cas9 technology in prostate cancer. Based on the rapid translation of this technology into the clinical application, it is anticipated that novel treatments based on this technique change the outcome of this malignancy in future.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Male
Humans
*CRISPR-Cas Systems/genetics
*Receptors, Androgen/genetics/metabolism
*Prostatic Neoplasms/genetics/metabolism/pathology
*Signal Transduction/genetics
Animals
RNA, Messenger/genetics/metabolism
RevDate: 2024-10-19
CmpDate: 2024-10-18
Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA.
Cell chemical biology, 31(10):1839-1851.e8.
A photocatalytic click chemistry approach, offering a significant advancement over conventional methods in RNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.
Additional Links: PMID-39383877
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39383877,
year = {2024},
author = {Qi, Q and Liu, X and Xiong, W and Zhang, K and Shen, W and Zhang, Y and Xu, X and Zhong, C and Zhang, Y and Tian, T and Zhou, X},
title = {Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA.},
journal = {Cell chemical biology},
volume = {31},
number = {10},
pages = {1839-1851.e8},
doi = {10.1016/j.chembiol.2024.09.006},
pmid = {39383877},
issn = {2451-9448},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *Gene Editing/methods ; Click Chemistry ; HEK293 Cells ; Light ; },
abstract = {A photocatalytic click chemistry approach, offering a significant advancement over conventional methods in RNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Humans
*Gene Editing/methods
Click Chemistry
HEK293 Cells
Light
RevDate: 2024-10-19
CmpDate: 2024-10-19
Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis.
Nature plants, 10(10):1502-1513.
Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants with variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, using new combinations of PE components, including an RNA chaperone and altered engineered prime editing guide RNAs driven by a PolII-PolIII composite promoter and a viral replicon system, we obtained up to 9.7% of the desired PE efficiency at the callus stage as assessed by targeted deep sequencing. Subsequently, we identified that up to 38.2% of transformants contained desired PE alleles in tomatoes and Arabidopsis, marking successful heritable PE transmission. Our PE tools also showed high accuracy, specificity and multiplexing capability, which unlocked the potential for practical PE applications in dicots, paving the way for transformative advancements in plant sciences.
Additional Links: PMID-39242983
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39242983,
year = {2024},
author = {Vu, TV and Nguyen, NT and Kim, J and Song, YJ and Nguyen, TH and Kim, JY},
title = {Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis.},
journal = {Nature plants},
volume = {10},
number = {10},
pages = {1502-1513},
pmid = {39242983},
issn = {2055-0278},
support = {2020M3A9I4038352//National Research Foundation of Korea (NRF)/ ; 2022R1A2C3010331//National Research Foundation of Korea (NRF)/ ; 2020R1A6A1A03044344//National Research Foundation of Korea (NRF)/ ; 2021R1A5A8029490//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Arabidopsis/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants with variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, using new combinations of PE components, including an RNA chaperone and altered engineered prime editing guide RNAs driven by a PolII-PolIII composite promoter and a viral replicon system, we obtained up to 9.7% of the desired PE efficiency at the callus stage as assessed by targeted deep sequencing. Subsequently, we identified that up to 38.2% of transformants contained desired PE alleles in tomatoes and Arabidopsis, marking successful heritable PE transmission. Our PE tools also showed high accuracy, specificity and multiplexing capability, which unlocked the potential for practical PE applications in dicots, paving the way for transformative advancements in plant sciences.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Solanum lycopersicum/genetics
*Arabidopsis/genetics
*Gene Editing/methods
Plants, Genetically Modified/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
RevDate: 2024-10-18
CmpDate: 2024-10-18
A line attractor encoding a persistent internal state requires neuropeptide signaling.
Cell, 187(21):5998-6015.e18.
Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1[+] neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.
Additional Links: PMID-39191257
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39191257,
year = {2024},
author = {Mountoufaris, G and Nair, A and Yang, B and Kim, DW and Vinograd, A and Kim, S and Linderman, SW and Anderson, DJ},
title = {A line attractor encoding a persistent internal state requires neuropeptide signaling.},
journal = {Cell},
volume = {187},
number = {21},
pages = {5998-6015.e18},
doi = {10.1016/j.cell.2024.08.015},
pmid = {39191257},
issn = {1097-4172},
mesh = {Animals ; *Neuropeptides/metabolism/genetics ; *Signal Transduction ; Mice ; Male ; Female ; *Neurons/metabolism ; CRISPR-Cas Systems/genetics ; Oxytocin/metabolism ; Hypothalamus/metabolism ; Gene Editing ; Receptors, Vasopressin/metabolism/genetics ; Mice, Inbred C57BL ; Estrogen Receptor alpha/metabolism ; },
abstract = {Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1[+] neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*Neuropeptides/metabolism/genetics
*Signal Transduction
Mice
Male
Female
*Neurons/metabolism
CRISPR-Cas Systems/genetics
Oxytocin/metabolism
Hypothalamus/metabolism
Gene Editing
Receptors, Vasopressin/metabolism/genetics
Mice, Inbred C57BL
Estrogen Receptor alpha/metabolism
RevDate: 2024-10-18
CmpDate: 2024-10-18
The Fanconi anemia pathway induces chromothripsis and ecDNA-driven cancer drug resistance.
Cell, 187(21):6055-6070.e22.
Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis.
Additional Links: PMID-39181133
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39181133,
year = {2024},
author = {Engel, JL and Zhang, X and Wu, M and Wang, Y and Espejo Valle-Inclán, J and Hu, Q and Woldehawariat, KS and Sanders, MA and Smogorzewska, A and Chen, J and Cortés-Ciriano, I and Lo, RS and Ly, P},
title = {The Fanconi anemia pathway induces chromothripsis and ecDNA-driven cancer drug resistance.},
journal = {Cell},
volume = {187},
number = {21},
pages = {6055-6070.e22},
doi = {10.1016/j.cell.2024.08.001},
pmid = {39181133},
issn = {1097-4172},
support = {R01 CA289435/CA/NCI NIH HHS/United States ; R01 GM140400/GM/NIGMS NIH HHS/United States ; R35 GM146610/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; *Chromothripsis ; *Fanconi Anemia/metabolism/genetics ; Fanconi Anemia Complementation Group Proteins/metabolism/genetics ; Mitosis ; Fanconi Anemia Complementation Group D2 Protein/metabolism/genetics ; CRISPR-Cas Systems/genetics ; DNA Replication ; Recombinases/metabolism ; DNA Repair ; Cell Line, Tumor ; Endonucleases/metabolism/genetics ; DNA Breaks, Double-Stranded ; Animals ; Mice ; Neoplasms/genetics/drug therapy/metabolism/pathology ; Ubiquitination ; },
abstract = {Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPR-Cas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCI-FANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces large-scale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extrachromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*Drug Resistance, Neoplasm/genetics
*Chromothripsis
*Fanconi Anemia/metabolism/genetics
Fanconi Anemia Complementation Group Proteins/metabolism/genetics
Mitosis
Fanconi Anemia Complementation Group D2 Protein/metabolism/genetics
CRISPR-Cas Systems/genetics
DNA Replication
Recombinases/metabolism
DNA Repair
Cell Line, Tumor
Endonucleases/metabolism/genetics
DNA Breaks, Double-Stranded
Animals
Mice
Neoplasms/genetics/drug therapy/metabolism/pathology
Ubiquitination
RevDate: 2024-10-19
CmpDate: 2024-10-19
A Novel CRISPR/Cas9-mediated Mouse Model of Colon Carcinogenesis.
Cellular and molecular gastroenterology and hepatology, 18(5):101390.
BACKGROUND & AIMS: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. The aim of this study was to generate a sporadic CRC model that more closely mimics this multi-step process and to use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis.
METHODS: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a doxycycline-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of 4 tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4, and Trp53. These were crossed to Vil-LCL-PLAGL2 mice, which have Cre-inducible overexpression of PLAGL2 in the gut epithelium.
RESULTS: These mice exhibited random somatic mutations in all 4 targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth.
CONCLUSIONS: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.
Additional Links: PMID-39128652
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39128652,
year = {2024},
author = {Kashima, H and Fischer, A and Veronese-Paniagua, DA and Gazit, VA and Ma, C and Yan, Y and Levin, MS and Madison, BB and Rubin, DC},
title = {A Novel CRISPR/Cas9-mediated Mouse Model of Colon Carcinogenesis.},
journal = {Cellular and molecular gastroenterology and hepatology},
volume = {18},
number = {5},
pages = {101390},
pmid = {39128652},
issn = {2352-345X},
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; Humans ; Carcinogenesis/genetics/pathology ; Colonic Neoplasms/genetics/pathology/metabolism ; Mice, Transgenic ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Transcription Factors/genetics/metabolism ; Mutation ; },
abstract = {BACKGROUND & AIMS: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. The aim of this study was to generate a sporadic CRC model that more closely mimics this multi-step process and to use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis.
METHODS: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a doxycycline-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of 4 tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4, and Trp53. These were crossed to Vil-LCL-PLAGL2 mice, which have Cre-inducible overexpression of PLAGL2 in the gut epithelium.
RESULTS: These mice exhibited random somatic mutations in all 4 targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth.
CONCLUSIONS: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Animals
*CRISPR-Cas Systems
Mice
*Disease Models, Animal
Humans
Carcinogenesis/genetics/pathology
Colonic Neoplasms/genetics/pathology/metabolism
Mice, Transgenic
Gene Expression Regulation, Neoplastic
Genes, Tumor Suppressor
Transcription Factors/genetics/metabolism
Mutation
RevDate: 2024-10-17
CmpDate: 2024-10-17
Intelligent guide RNA: dual toehold switches for modulating luciferase in the presence of trigger RNA.
Communications biology, 7(1):1344.
The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA-a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs-is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform.
Additional Links: PMID-39420075
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39420075,
year = {2024},
author = {Hashemabadi, M and Sasan, HA and Hosseinkhani, S and Amandadi, M and Samareh Gholami, A and Sadeghizadeh, M},
title = {Intelligent guide RNA: dual toehold switches for modulating luciferase in the presence of trigger RNA.},
journal = {Communications biology},
volume = {7},
number = {1},
pages = {1344},
pmid = {39420075},
issn = {2399-3642},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *Luciferases/genetics/metabolism ; Humans ; Genes, Reporter ; Synthetic Biology/methods ; },
abstract = {The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA-a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs-is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems
*Luciferases/genetics/metabolism
Humans
Genes, Reporter
Synthetic Biology/methods
RevDate: 2024-10-17
CmpDate: 2024-10-17
CRISPRi-mediated metabolic switch enables concurrent aerobic and synthetic anaerobic fermentations in engineered consortium.
Nature communications, 15(1):8985.
Replacing petrochemicals with compounds from bio-based manufacturing processes remains an important part of the global effort to move towards a sustainable future. However, achieving economic viability requires both optimized cell factories and innovative processes. Here, we address this challenge by developing a fermentation platform, which enables two concurrent fermentations in one bioreactor. We first construct a xylitol producing Escherichia coli strain in which CRISPRi-mediated gene silencing is used to switch the metabolism from aerobic to anaerobic, even when the bacteria are under oxic conditions. The switch also decouples growth from production, which further increases the yield. The strain produces acetate as a byproduct, which is subsequently metabolized under oxic conditions by a secondary E. coli strain. Through constraint-based metabolic modelling this strain is designed to co-valorize glucose and the excreted acetate to a secondary product. This unique syntrophic consortium concept facilitates the implementation of "two fermentations in one go", where the concurrent fermentation displays similar titers and productivities as compared to two separate single strain fermentations.
Additional Links: PMID-39420027
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39420027,
year = {2024},
author = {Rong, Y and Frey, A and Özdemir, E and Sainz de la Maza Larrea, A and Li, S and Nielsen, AT and Jensen, SI},
title = {CRISPRi-mediated metabolic switch enables concurrent aerobic and synthetic anaerobic fermentations in engineered consortium.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8985},
pmid = {39420027},
issn = {2041-1723},
support = {NNF20CC0035580//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF17SA0031362//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; NNF21SA0069783//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; 7017-00321B//Det Frie Forskningsråd (Danish Council for Independent Research)/ ; },
mesh = {*Fermentation ; Anaerobiosis ; *Escherichia coli/metabolism/genetics ; *Metabolic Engineering/methods ; Aerobiosis ; *Bioreactors/microbiology ; Acetates/metabolism ; Glucose/metabolism ; Xylitol/metabolism ; CRISPR-Cas Systems ; },
abstract = {Replacing petrochemicals with compounds from bio-based manufacturing processes remains an important part of the global effort to move towards a sustainable future. However, achieving economic viability requires both optimized cell factories and innovative processes. Here, we address this challenge by developing a fermentation platform, which enables two concurrent fermentations in one bioreactor. We first construct a xylitol producing Escherichia coli strain in which CRISPRi-mediated gene silencing is used to switch the metabolism from aerobic to anaerobic, even when the bacteria are under oxic conditions. The switch also decouples growth from production, which further increases the yield. The strain produces acetate as a byproduct, which is subsequently metabolized under oxic conditions by a secondary E. coli strain. Through constraint-based metabolic modelling this strain is designed to co-valorize glucose and the excreted acetate to a secondary product. This unique syntrophic consortium concept facilitates the implementation of "two fermentations in one go", where the concurrent fermentation displays similar titers and productivities as compared to two separate single strain fermentations.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*Fermentation
Anaerobiosis
*Escherichia coli/metabolism/genetics
*Metabolic Engineering/methods
Aerobiosis
*Bioreactors/microbiology
Acetates/metabolism
Glucose/metabolism
Xylitol/metabolism
CRISPR-Cas Systems
RevDate: 2024-10-17
CmpDate: 2024-10-17
Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness.
Nature communications, 15(1):8966.
Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.
Additional Links: PMID-39419994
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39419994,
year = {2024},
author = {Rosen, BP and Li, QV and Cho, HS and Liu, D and Yang, D and Graff, S and Yan, J and Luo, R and Verma, N and Damodaran, JR and Kale, HT and Kaplan, SJ and Beer, MA and Sidoli, S and Huangfu, D},
title = {Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {8966},
pmid = {39419994},
issn = {2041-1723},
support = {U01HG012051//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; UM1HG012654//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG012051//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG012051//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; *Cell Differentiation/genetics ; Chromatin/metabolism/genetics ; Genome, Human ; Cell Self Renewal/genetics ; },
abstract = {Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
Humans
*CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism/cytology
*Cell Differentiation/genetics
Chromatin/metabolism/genetics
Genome, Human
Cell Self Renewal/genetics
RevDate: 2024-10-17
Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems.
bioRxiv : the preprint server for biology pii:2024.10.09.617463.
Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target the RAI1 gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intact RAI1 copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.
Additional Links: PMID-39416069
Full Text:
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39416069,
year = {2024},
author = {Katz, N and An, C and Lee, YJ and Tycko, J and Zhang, M and Kang, J and Bintu, L and Bassik, MC and Huang, WH and Gao, XJ},
title = {Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.10.09.617463},
pmid = {39416069},
issn = {2692-8205},
abstract = {Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target the RAI1 gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intact RAI1 copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.},
}
RevDate: 2024-10-18
CmpDate: 2024-10-18
Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.
ACS synthetic biology, 13(10):3212-3230.
The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from trans cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.
Additional Links: PMID-39377757
Publisher:
PubMed:
Citation:
show bibtex listing
hide bibtex listing
@article {pmid39377757,
year = {2024},
author = {Wan, Y and Helenek, C and Coraci, D and Balázsi, G},
title = {Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.},
journal = {ACS synthetic biology},
volume = {13},
number = {10},
pages = {3212-3230},
doi = {10.1021/acssynbio.4c00271},
pmid = {39377757},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Regulatory Networks ; HEK293 Cells ; RNA/genetics ; Down-Regulation/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from trans cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.},
}
MeSH Terms:
show MeSH Terms
hide MeSH Terms
*CRISPR-Cas Systems/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Regulatory Networks
HEK293 Cells
RNA/genetics
Down-Regulation/genetics
CRISPR-Associated Proteins/genetics/metabolism
Gene Editing/methods
▼ ▼ LOAD NEXT 100 CITATIONS
ESP Quick Facts
ESP Origins
In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.
ESP Support
In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.
ESP Rationale
Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.
ESP Goal
In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.
ESP Usage
Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.
ESP Content
When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.
ESP Help
Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.
ESP Plans
With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.
ESP Picks from Around the Web (updated 28 JUL 2024 )
Old Science
Weird Science
Treating Disease with Fecal Transplantation
Fossils of miniature humans (hobbits) discovered in Indonesia
Paleontology
Dinosaur tail, complete with feathers, found preserved in amber.
Astronomy
Mysterious fast radio burst (FRB) detected in the distant universe.
Big Data & Informatics
Big Data: Buzzword or Big Deal?
Hacking the genome: Identifying anonymized human subjects using publicly available data.