@article {pmid38627378,
year = {2024},
author = {Wang, Y and Chen, H and Lin, K and Han, Y and Gu, Z and Wei, H and Mu, K and Wang, D and Liu, L and Jin, R and Song, R and Rong, Z and Wang, S},
title = {Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3279},
pmid = {38627378},
issn = {2041-1723},
mesh = {Humans ; Male ; *Monkeypox virus ; Biological Assay ; Cell Death ; *Exanthema ; Nucleotidyltransferases ; Nucleic Acid Amplification Techniques ; Sensitivity and Specificity ; CRISPR-Cas Systems ; Recombinases ; },
abstract = {The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive detection of MPXV in resource-limited settings. The viral nucleic acids are rapidly released from the rash fluid swab, oral swab, saliva, and urine samples in 2 min via a streamlined viral lysis protocol, followed by a 10-min single-step recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves the whole process for reaction execution, signal acquisition, and result interpretation. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer. We validate the developed assay on 102 clinical samples from male patients / volunteers, and the testing results are 100% concordant with the real-time PCR. SCOPE achieves a single-molecular level sensitivity in minutes with a simplified procedure performed on a miniaturized wireless device, which is expected to spur substantial progress to enable the practice application of CRISPR-based diagnostics techniques in a point-of-care setting.},
}

Humans
Male
*Monkeypox virus
Biological Assay
Cell Death
*Exanthema
Nucleotidyltransferases
Nucleic Acid Amplification Techniques
Sensitivity and Specificity
CRISPR-Cas Systems
Recombinases

@article {pmid38623982,
year = {2024},
author = {Volodina, OV and Fabrichnikova, AR and Anuchina, AA and Mishina, OS and Lavrov, AV and Smirnikhina, SA},
title = {Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232295117240405070809},
pmid = {38623982},
issn = {1875-5631},
abstract = {The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.},
}

@article {pmid38514901,
year = {2024},
author = {Zhai, D and Li, L and Chen, C and Wang, X and Liu, R and Shan, Y},
title = {INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.},
journal = {Journal of clinical laboratory analysis},
volume = {38},
number = {7},
pages = {e25031},
pmid = {38514901},
issn = {1098-2825},
mesh = {*Cilia/metabolism ; *Phosphoric Monoester Hydrolases/metabolism/genetics ; Humans ; Cell Line ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Retinal Pigment Epithelium/metabolism/cytology ; Phosphatidylinositol Phosphates/metabolism ; CRISPR-Cas Systems ; Phospholipids/metabolism ; },
abstract = {BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function.

METHODS: The EGFP-2xP4M[SidM], PH[PLCδ1]-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ.

RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP.

CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.},
}

*Cilia/metabolism
*Phosphoric Monoester Hydrolases/metabolism/genetics
Humans
Cell Line
Phosphatidylinositol 4,5-Diphosphate/metabolism
Retinal Pigment Epithelium/metabolism/cytology
Phosphatidylinositol Phosphates/metabolism
CRISPR-Cas Systems
Phospholipids/metabolism

@article {pmid38355914,
year = {2024},
author = {McLaurin, KA and Li, H and Khalili, K and Mactutus, CF and Booze, RM},
title = {HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.},
journal = {Journal of neurovirology},
volume = {30},
number = {1},
pages = {71-85},
pmid = {38355914},
issn = {1538-2443},
support = {DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; DA059310/DA/NIDA NIH HHS/United States ; DA056288/DA/NIDA NIH HHS/United States ; MH106392/MH/NIMH NIH HHS/United States ; NS100624/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *HIV-1/genetics/physiology ; Rats ; *CRISPR-Cas Systems ; *Rats, Transgenic ; *RNA, Messenger/genetics/metabolism ; *Gene Editing/methods ; Neuroglia/virology/metabolism ; Dependovirus/genetics ; HIV Infections/virology/genetics ; Gene Knockdown Techniques ; RNA, Viral/genetics ; Cognition/physiology ; Humans ; },
abstract = {Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 10[9], 8.46 × 10[9], 1.269 × 10[10], 1.692 × 10[10], 2.115 × 10[10], and 2.538 × 10[10] gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.},
}

Animals
*HIV-1/genetics/physiology
Rats
*CRISPR-Cas Systems
*Rats, Transgenic
*RNA, Messenger/genetics/metabolism
*Gene Editing/methods
Neuroglia/virology/metabolism
Dependovirus/genetics
HIV Infections/virology/genetics
Gene Knockdown Techniques
RNA, Viral/genetics
Cognition/physiology
Humans

@article {pmid38624901,
year = {2022},
author = {Mondal, S and Halder, SK and Mondal, KC},
title = {Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system.},
journal = {Systems microbiology and biomanufacturing},
volume = {2},
number = {1},
pages = {113-129},
pmid = {38624901},
issn = {2662-7663},
abstract = {Cellulose is the utmost plenteous source of biopolymer in our earth, and fungi are the most efficient and ubiquitous organism in degrading the cellulosic biomass by synthesizing cellulases. Tailoring through genetic manipulation has played a substantial role in constructing novel fungal strains towards improved cellulase production of desired traits. However, the traditional methods of genetic manipulation of fungi are time-consuming and tedious. With the availability of the full-genome sequences of several industrially relevant filamentous fungi, CRISPR-CAS (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) technology has come into the focus for the proficient development of manipulated strains of filamentous fungi. This review summarizes the mode of action of cellulases, transcription level regulation for cellulase expression, various traditional strategies of genetic manipulation with CRISPR-CAS technology to develop modified fungal strains for a preferred level of cellulase production, and the futuristic trend in this arena of research.},
}

@article {pmid38622798,
year = {2024},
author = {Peng, W and Gao, M and Zhu, X and Liu, X and Yang, G and Li, S and Liu, Y and Bai, L and Yang, J and Bao, J},
title = {Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.},
journal = {Biotechnology journal},
volume = {19},
number = {4},
pages = {e2300691},
doi = {10.1002/biot.202300691},
pmid = {38622798},
issn = {1860-7314},
support = {82270662//National Natural Science Foundation of China/ ; 82070640//National Natural Science Foundation of China/ ; 2022-YF05-01255-SN//Technology Innovation R&D Project of Chengdu Science and Technology Bureau/ ; 22ZDYF1406//Key R&D Project of Sichuan Science and Technology Department/ ; ZYJC21014//1.3.5 project for disciplines of excellence, West China Hospital Sichuan University/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knockout Techniques ; Genome ; RNA, Messenger/genetics ; },
abstract = {CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the β4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Gene Knockout Techniques
Genome
RNA, Messenger/genetics

@article {pmid38622404,
year = {2024},
author = {Freen-van Heeren, JJ},
title = {Employing CRISPR-Cas9 to Enhance T Cell Effector Function.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {195-208},
pmid = {38622404},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Gene Editing/methods ; Genetic Engineering/methods ; Cytokines/genetics ; },
abstract = {As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Gene Editing/methods
Genetic Engineering/methods
Cytokines/genetics

@article {pmid38622403,
year = {2024},
author = {Liao, X and Li, L},
title = {CRISPR-Cas9-Induced Gene Editing in Primary Human Monocytes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2782},
number = {},
pages = {189-193},
pmid = {38622403},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; Monocytes/metabolism ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Monocytes play important and diverse roles in both homeostatic and inflammatory immune responses. The CRISPR-Cas9 system in lentiviral vectors has been widely used to manipulate specific genes of immortal monocyte cell lines to study monocyte functions. However, human primary monocytes are refractory to this method with low gene knockout (KO) efficiency. In this chapter, we developed an in vitro gene-editing procedure for primary human monocytes with a consistent and high-gene KO efficiency via a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single-guide RNA (sgRNA). This method can be adapted to study the functions of targeted signaling molecules involved in modulating monocyte polarization in primary human monocytes.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
Monocytes/metabolism
CRISPR-Associated Protein 9/genetics

@article {pmid38622379,
year = {2024},
author = {Prostova, M and Kanevskaya, A and Panteleev, V and Lisitskaya, L and Perfilova Tugaeva, KV and Sluchanko, NN and Esyunina, D and Kulbachinskiy, A},
title = {DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {38622379},
issn = {2058-5276},
support = {22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; 22-14-00182//Russian Science Foundation (RSF)/ ; },
abstract = {Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.},
}

@article {pmid38622229,
year = {2024},
author = {Xiang, X and Yang, H and Yuan, X and Dong, X and Mai, S and Zhang, Q and Chen, L and Cao, D and Chen, H and Guo, W and Li, L},
title = {CRISPR/Cas9-mediated editing of GmDWF1 brassinosteroid biosynthetic gene induces dwarfism in soybean.},
journal = {Plant cell reports},
volume = {43},
number = {5},
pages = {116},
pmid = {38622229},
issn = {1432-203X},
support = {32071926//The National Natural Science Foundation of China/ ; 32072087//The National Natural Science Foundation of China/ ; CAAS-OCRI-XKPY-202103//Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Brassinosteroids/metabolism ; Glycine max/genetics ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; *Arabidopsis/metabolism ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; },
abstract = {The study on the GmDWF1-deficient mutant dwf1 showed that GmDWF1 plays a crucial role in determining soybean plant height and yield by influencing the biosynthesis of brassinosteroids. Soybean has not adopted the Green Revolution, such as reduced height for increased planting density, which have proven beneficial for cereal crops. Our research identified the soybean genes GmDWF1a and GmDWF1b, homologous to Arabidopsis AtDWF1, and found that they are widely expressed, especially in leaves, and linked to the cellular transport system, predominantly within the endoplasmic reticulum and intracellular vesicles. These genes are essential for the synthesis of brassinosteroids (BR). Single mutants of GmDWF1a and GmDWF1b, as well as double mutants of both genes generated through CRISPR/Cas9 genome editing, exhibit a dwarf phenotype. The single-gene mutant exhibits moderate dwarfism, while the double mutant shows more pronounced dwarfism. Despite the reduced stature, all types of mutants preserve their node count. Notably, field tests have shown that the single GmDWF1a mutant produced significantly more pods than wild-type plants. Spraying exogenous brassinolide (BL) can compensate for the loss in plant height induced by the decrease in endogenous BRs. Comparing transcriptome analyses of the GmDWF1a mutant and wild-type plants revealed a significant impact on the expression of many genes that influence soybean growth. Identifying the GmDWF1a and GmDWF1b genes could aid in the development of compact, densely planted soybean varieties, potentially boosting productivity.},
}

*Brassinosteroids/metabolism
Glycine max/genetics
CRISPR-Cas Systems/genetics
Mutation/genetics
*Arabidopsis/metabolism
Gene Editing
Gene Expression Regulation, Plant/genetics

@article {pmid38622170,
year = {2024},
author = {Lee, SH and Wang, CY and Li, IJ and Abe, G and Ota, KG},
title = {Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8716},
pmid = {38622170},
issn = {2045-2322},
support = {112-2311-B-001-033//National Science and Technology Council/ ; },
mesh = {Animals ; *Goldfish/genetics ; Alleles ; *CRISPR-Cas Systems ; Biological Evolution ; Mutation ; Phenotype ; Animals, Domestic/genetics ; },
abstract = {Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdA[E127X])-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.},
}

Animals
*Goldfish/genetics
Alleles
*CRISPR-Cas Systems
Biological Evolution
Mutation
Phenotype
Animals, Domestic/genetics

@article {pmid38621214,
year = {2024},
author = {Srivastava, R and Davison, CW and Krull, AG and Entriken, SM and Zumbrock, A and Cortes Hidalgo, MD and Adair, KJ and Escherich, AM and Lara, JN and Neverman, EC and Hodnefield, M and McElligtot, E and Sandquist, EJ and Ogilvie, C and Lafontant, P and Essner, JJ},
title = {An Undergraduate Course in CRISPR/Cas9-Mediated Gene Editing in Zebrafish.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {162-170},
doi = {10.1089/zeb.2023.0091},
pmid = {38621214},
issn = {1557-8542},
mesh = {Humans ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Students ; },
abstract = {We have developed a one-credit semester-long research experience for undergraduate students that involves the use of CRISPR/Cas9 to edit genes in zebrafish. The course is available to students at all stages of their undergraduate training and can be taken up to four times. Students select a gene of interest to edit as the basis of their semester-long project. To select a gene, exploration of developmental processes and human disease is encouraged. As part of the course, students use basic bioinformatic tools, design guide RNAs, inject zebrafish embryos, and analyze both the molecular consequences of gene editing and phenotypic outcomes. Over the 10 years we have offered the course, enrollment has grown from less than 10 students to more than 60 students per semester. Each year, we choose a different gene editing strategy to explore based on recent publications of gene editing methodologies. These have included making CRISPants, targeted integrations, and large gene deletions. In this study, we present how we structure the course and our assessment of the course over the past 3 years.},
}

Humans
Animals
*Gene Editing/methods
*CRISPR-Cas Systems
Zebrafish/genetics
RNA, Guide, CRISPR-Cas Systems
Students

@article {pmid38621205,
year = {2024},
author = {Davison, C and Harzman, H and Nicholson, J and Entriken, S and Mobley, K and Krull, A and Singhal, M and Skow, C and Matthews, N and Kopp, L and Gillette, B and Weide, TJ and Hukvari, JR and Stumpf, SCP and Feldmann, OM and McGrail, M and Srivastava, R and Essner, JJ},
title = {Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms.},
journal = {Zebrafish},
volume = {21},
number = {2},
pages = {191-197},
doi = {10.1089/zeb.2023.0096},
pmid = {38621205},
issn = {1557-8542},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; Red Fluorescent Protein ; Biotin/genetics ; Endothelial Cells ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFP[is86] line to examine the behavior and interactions between cells during vascular formation in zebrafish.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
Red Fluorescent Protein
Biotin/genetics
Endothelial Cells
RNA, Guide, CRISPR-Cas Systems

@article {pmid38616491,
year = {2024},
author = {Tang, X and Ren, Q and Yan, X and Zhang, R and Liu, L and Han, Q and Zheng, X and Qi, Y and Song, H and Zhang, Y},
title = {Boosting genome editing in plants with single transcript unit surrogate reporter systems.},
journal = {Plant communications},
volume = {},
number = {},
pages = {100921},
doi = {10.1016/j.xplc.2024.100921},
pmid = {38616491},
issn = {2590-3462},
abstract = {CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of edited events. In this study, we introduce multiple Single Transcript Unit Surrogate Reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems utilize the same sgRNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are employed to restore functional reporter genes post-genome editing, including efficient single strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-BE systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are demonstrated to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.},
}

@article {pmid38583236,
year = {2024},
author = {Wong, BL and Mendoza, HG and Jacobsen, CS and Beal, PA},
title = {RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.},
journal = {Bioorganic & medicinal chemistry},
volume = {104},
number = {},
pages = {117700},
doi = {10.1016/j.bmc.2024.117700},
pmid = {38583236},
issn = {1464-3391},
mesh = {Humans ; Adenosine ; *Adenosine Deaminase/metabolism ; Base Sequence ; *Purine Nucleosides ; *Ribonucleosides ; RNA, Double-Stranded ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.},
}

Humans
Adenosine
*Adenosine Deaminase/metabolism
Base Sequence
*Purine Nucleosides
*Ribonucleosides
RNA, Double-Stranded
RNA, Guide, CRISPR-Cas Systems

@article {pmid38527624,
year = {2024},
author = {Piñón Hofbauer, J and Guttmann-Gruber, C and Wally, V and Sharma, A and Gratz, IK and Koller, U},
title = {Challenges and progress related to gene editing in rare skin diseases.},
journal = {Advanced drug delivery reviews},
volume = {208},
number = {},
pages = {115294},
doi = {10.1016/j.addr.2024.115294},
pmid = {38527624},
issn = {1872-8294},
mesh = {Humans ; *Gene Editing ; CRISPR-Cas Systems/genetics ; Quality of Life ; Skin ; *Skin Diseases/genetics/therapy ; },
abstract = {Genodermatoses represent a large group of inherited skin disorders encompassing clinically-heterogeneous conditions that manifest in the skin and other organs. Depending on disease variant, associated clinical manifestations and secondary complications can severely impact patients' quality of life and currently available treatments are transient and not curative. Multiple emerging approaches using CRISPR-based technologies offer promising prospects for therapy. Here, we explore current advances and challenges related to gene editing in rare skin diseases, including different strategies tailored to mutation type and structural organization of the affected gene, considerations for in vivo and ex vivo applications, the critical issue of delivery into the skin, and immune aspects of therapy. Against the backdrop of a landmark FDA approval for the first re-dosable gene replacement therapy for a rare genetic skin disorder, gene editing approaches are inching closer to the clinics and the possibility of a local permanent cure for patients affected by these disorders.},
}

Humans
*Gene Editing
CRISPR-Cas Systems/genetics
Quality of Life
Skin
*Skin Diseases/genetics/therapy

@article {pmid38517011,
year = {2024},
author = {Lau, MSH and Madika, A and Zhang, Y and Minton, NP},
title = {Parageobacillus thermoglucosidasius Strain Engineering Using a Theophylline Responsive RiboCas for Controlled Gene Expression.},
journal = {ACS synthetic biology},
volume = {13},
number = {4},
pages = {1237-1245},
doi = {10.1021/acssynbio.3c00735},
pmid = {38517011},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Theophylline ; Gene Editing ; Gene Expression ; *Bacillaceae ; },
abstract = {The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.},
}

*CRISPR-Cas Systems/genetics
*Theophylline
Gene Editing
Gene Expression
*Bacillaceae

@article {pmid38508753,
year = {2024},
author = {Carvajal-Moreno, J and Wang, X and Hernandez, VA and Mondal, M and Zhao, X and Yalowich, JC and Elton, TS},
title = {Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβ in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.},
journal = {The Journal of pharmacology and experimental therapeutics},
volume = {389},
number = {2},
pages = {186-196},
doi = {10.1124/jpet.123.002038},
pmid = {38508753},
issn = {1521-0103},
mesh = {Humans ; Etoposide/pharmacology ; K562 Cells ; DNA Topoisomerases, Type II/genetics/metabolism ; Mitoxantrone ; CRISPR-Cas Systems/genetics ; Codon, Nonsense ; *Antineoplastic Agents/pharmacology ; *Leukemia ; DNA ; Phenotype ; },
abstract = {DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2β/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2β/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2β/180, a premature stop codon was generated at the TOP2β/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2β/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2β/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2β/180 gene-edited clones, with one or all four TOP2β/180 alleles mutated, revealed partial or complete loss of TOP2β mRNA/protein, respectively. The loss of TOP2β/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}p
ropionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2β/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.},
}

Humans
Etoposide/pharmacology
K562 Cells
DNA Topoisomerases, Type II/genetics/metabolism
Mitoxantrone
CRISPR-Cas Systems/genetics
Codon, Nonsense
*Antineoplastic Agents/pharmacology
*Leukemia
DNA
Phenotype

@article {pmid38493007,
year = {2024},
author = {Dimitrievska, M and Bansal, D and Vitale, M and Strouboulis, J and Miccio, A and Nicolaides, KH and El Hoss, S and Shangaris, P and Jacków-Malinowska, J},
title = {Revolutionising healing: Gene Editing's breakthrough against sickle cell disease.},
journal = {Blood reviews},
volume = {65},
number = {},
pages = {101185},
doi = {10.1016/j.blre.2024.101185},
pmid = {38493007},
issn = {1532-1681},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems ; *Anemia, Sickle Cell/genetics/therapy ; *Hemoglobinopathies/genetics ; Fetal Hemoglobin/genetics ; },
abstract = {Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the β-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD.},
}

Humans
Gene Editing/methods
CRISPR-Cas Systems
*Anemia, Sickle Cell/genetics/therapy
*Hemoglobinopathies/genetics
Fetal Hemoglobin/genetics

@article {pmid38479385,
year = {2024},
author = {Ma, X and Yin, J and Qiao, L and Wan, H and Liu, X and Zhou, Y and Wu, J and Niu, L and Wu, M and Wang, X and Ye, H},
title = {A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.},
journal = {Molecular cell},
volume = {84},
number = {8},
pages = {1585-1600.e7},
doi = {10.1016/j.molcel.2024.02.019},
pmid = {38479385},
issn = {1097-4164},
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; CRISPR-Associated Protein 9/genetics/metabolism ; Proteins/metabolism ; Proteolysis ; Mammals/metabolism ; },
abstract = {Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.},
}

Mice
Animals
*CRISPR-Cas Systems
*Gene Editing
CRISPR-Associated Protein 9/genetics/metabolism
Proteins/metabolism
Proteolysis
Mammals/metabolism

@article {pmid38460553,
year = {2024},
author = {Ren, QW and Liu, TY and Lan, HJ and Li, ZC and Huang, MJ and Zhao, YT and Chen, Y and Liao, LN and Ma, XH and Liu, JZ},
title = {Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {343},
number = {},
pages = {112057},
doi = {10.1016/j.plantsci.2024.112057},
pmid = {38460553},
issn = {1873-2259},
mesh = {*Nicotiana/genetics ; *Arabidopsis/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; Protein Kinases/genetics ; Plants/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.},
}

*Nicotiana/genetics
*Arabidopsis/genetics/metabolism
Indoleacetic Acids/metabolism
CRISPR-Cas Systems
Protein Kinases/genetics
Plants/metabolism
Gene Expression Regulation, Plant

@article {pmid38613390,
year = {2024},
author = {Cardiff, RAL and Faulkner, ID and Beall, JG and Carothers, JM and Zalatan, JG},
title = {CRISPR-Cas tools for simultaneous transcription & translation control in bacteria.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkae275},
pmid = {38613390},
issn = {1362-4962},
support = {MCB 2225632//National Science Foundation/ ; },
abstract = {Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.},
}

@article {pmid38612794,
year = {2024},
author = {Stahl, F and Evert, BO and Han, X and Breuer, P and Wüllner, U},
title = {Spinocerebellar Ataxia Type 3 Pathophysiology-Implications for Translational Research and Clinical Studies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {7},
pages = {},
pmid = {38612794},
issn = {1422-0067},
mesh = {Humans ; Animals ; *Machado-Joseph Disease/genetics ; Translational Research, Biomedical ; *Spinocerebellar Ataxias/genetics ; Translational Science, Biomedical ; Animals, Genetically Modified ; },
abstract = {The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.},
}

Humans
Animals
*Machado-Joseph Disease/genetics
Translational Research, Biomedical
*Spinocerebellar Ataxias/genetics
Translational Science, Biomedical
Animals, Genetically Modified

@article {pmid38609947,
year = {2024},
author = {Im, SH and Jang, M and Park, JH and Chung, HJ},
title = {Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.},
journal = {Journal of nanobiotechnology},
volume = {22},
number = {1},
pages = {175},
pmid = {38609947},
issn = {1477-3155},
support = {2021R1A2C2011763//National Research Foundation of Korea/ ; 2021R1A2C2011763//National Research Foundation of Korea/ ; HI22C2010//Ministry of Health and Welfare/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Cell Line ; Ribonucleoproteins/genetics ; *Liposomes ; *Nanoparticles ; },
abstract = {Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.},
}

*Gene Editing
*CRISPR-Cas Systems
Cell Line
Ribonucleoproteins/genetics
*Liposomes
*Nanoparticles

@article {pmid38609863,
year = {2024},
author = {Chen, X and Zhong, S and Zhan, Y and Zhang, X},
title = {CRISPR-Cas9 applications in T cells and adoptive T cell therapies.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {52},
pmid = {38609863},
issn = {1689-1392},
support = {82203862//National Natural Science Foundation of China/ ; 222300420344//Natural Science Foundation of Henan Province/ ; SBGJ202102140//Medical Science Technology Program of Henan/ ; LHGJ20200273//Medical Science Technology Program of Henan/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes ; Cell Differentiation ; Inflammation ; Lymphocyte Activation ; },
abstract = {T cell immunity is central to contemporary cancer and autoimmune therapies, encompassing immune checkpoint blockade and adoptive T cell therapies. Their diverse characteristics can be reprogrammed by different immune challenges dependent on antigen stimulation levels, metabolic conditions, and the degree of inflammation. T cell-based therapeutic strategies are gaining widespread adoption in oncology and treating inflammatory conditions. Emerging researches reveal that clustered regularly interspaced palindromic repeats-associated protein 9 (CRISPR-Cas9) genome editing has enabled T cells to be more adaptable to specific microenvironments, opening the door to advanced T cell therapies in preclinical and clinical trials. CRISPR-Cas9 can edit both primary T cells and engineered T cells, including CAR-T and TCR-T, in vivo and in vitro to regulate T cell differentiation and activation states. This review first provides a comprehensive summary of the role of CRISPR-Cas9 in T cells and its applications in preclinical and clinical studies for T cell-based therapies. We also explore the application of CRISPR screen high-throughput technology in editing T cells and anticipate the current limitations of CRISPR-Cas9, including off-target effects and delivery challenges, and envisioned improvements in related technologies for disease screening, diagnosis, and treatment.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes
Cell Differentiation
Inflammation
Lymphocyte Activation

@article {pmid38609574,
year = {2024},
author = {De Castro, V and Galaine, J and Loyon, R and Godet, Y},
title = {CRISPR-Cas gene knockouts to optimize engineered T cells for cancer immunotherapy.},
journal = {Cancer gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38609574},
issn = {1476-5500},
support = {2022-0047//Ligue Contre le Cancer/ ; },
abstract = {While CAR-T and tgTCR-T therapies have exhibited noteworthy and promising outcomes in hematologic and solid tumors respectively, a set of distinct challenges remains. Consequently, the quest for novel strategies has become imperative to safeguard and more effectively release the full functions of engineered T cells. These factors are intricately linked to the success of adoptive cell therapy. Recently, CRISPR-based technologies have emerged as a major breakthrough for maintaining T cell functions. These technologies have allowed the discovery of T cells' negative regulators such as specific cell-surface receptors, cell-signaling proteins, and transcription factors that are involved in the development or maintenance of T cell dysfunction. By employing a CRISPR-genic invalidation approach to target these negative regulators, it has become possible to prevent the emergence of hypofunctional T cells. This review revisits the establishment of the dysfunctional profile of T cells before delving into a comprehensive summary of recent CRISPR-gene invalidations, with each invalidation contributing to the enhancement of engineered T cells' antitumor capacities. The narrative unfolds as we explore how these advancements were discovered and identified, marking a significant advancement in the pursuit of superior adoptive cell therapy.},
}

@article {pmid38609487,
year = {2024},
author = {Naveed, M and Tahir, F and Aziz, T and Waseem, M and Makhdoom, SI and Ali, N and Alharbi, M and Albekairi, TH and Alasmari, AF},
title = {Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8563},
pmid = {38609487},
issn = {2045-2322},
mesh = {Humans ; *Cholera ; Proteus mirabilis/genetics ; Cadmium/toxicity ; CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S ; Wastewater ; RNA, Guide, CRISPR-Cas Systems ; *Vibrio/genetics ; *Urinary Tract Infections ; },
abstract = {Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl2. 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.},
}

Humans
*Cholera
Proteus mirabilis/genetics
Cadmium/toxicity
CRISPR-Cas Systems/genetics
RNA, Ribosomal, 16S
Wastewater
RNA, Guide, CRISPR-Cas Systems
*Vibrio/genetics
*Urinary Tract Infections

@article {pmid38609352,
year = {2024},
author = {McLean, ZL and Gao, D and Correia, K and Roy, JCL and Shibata, S and Farnum, IN and Valdepenas-Mellor, Z and Kovalenko, M and Rapuru, M and Morini, E and Ruliera, J and Gillis, T and Lucente, D and Kleinstiver, BP and Lee, JM and MacDonald, ME and Wheeler, VC and Mouro Pinto, R and Gusella, JF},
title = {Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {3182},
pmid = {38609352},
issn = {2041-1723},
support = {NS091161//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS105709//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS119471//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS049206//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; NS126420//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; DP2-CA281401//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Huntington Disease/genetics ; Exons/genetics ; Gene Expression Profiling ; Heterozygote ; Homozygote ; MutL Proteins ; Neoplasm Proteins ; },
abstract = {Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.},
}

Humans
*Huntington Disease/genetics
Exons/genetics
Gene Expression Profiling
Heterozygote
Homozygote
MutL Proteins
Neoplasm Proteins

@article {pmid38579141,
year = {2024},
author = {Moon, SY and Zhang, D and Chen, SC and Lamey, TM and Thompson, JA and McLaren, TL and Chen, FK and McLenachan, S},
title = {Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {100-110},
doi = {10.1089/crispr.2023.0065},
pmid = {38579141},
issn = {2573-1602},
mesh = {Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Reactive Oxygen Species/metabolism ; Virulence ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Eye Proteins/genetics/metabolism ; Membrane Proteins/genetics ; Nerve Tissue Proteins/genetics/metabolism ; },
abstract = {Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.},
}

Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Reactive Oxygen Species/metabolism
Virulence
*CRISPR-Cas Systems
Gene Editing
Gene Expression
Eye Proteins/genetics/metabolism
Membrane Proteins/genetics
Nerve Tissue Proteins/genetics/metabolism

@article {pmid38578788,
year = {2024},
author = {Ma, S and Ni, X and Chen, S and Qiao, X and Xu, X and Chen, W and Champer, J and Huang, J},
title = {A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.},
journal = {PLoS genetics},
volume = {20},
number = {4},
pages = {e1011226},
pmid = {38578788},
issn = {1553-7404},
mesh = {Female ; Animals ; Humans ; Drosophila/genetics ; Drosophila melanogaster/genetics ; *Gene Drive Technology ; *Infertility, Female/genetics ; CRISPR-Cas Systems ; Fruit ; RNA, Guide, CRISPR-Cas Systems ; Phenotype ; },
abstract = {CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.},
}

Female
Animals
Humans
Drosophila/genetics
Drosophila melanogaster/genetics
*Gene Drive Technology
*Infertility, Female/genetics
CRISPR-Cas Systems
Fruit
RNA, Guide, CRISPR-Cas Systems
Phenotype

@article {pmid38564198,
year = {2024},
author = {Levrier, G},
title = {Making the Search for Genome Editing Tortured Phrases a Collective Effort.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {72},
doi = {10.1089/crispr.2024.0015},
pmid = {38564198},
issn = {2573-1602},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; },
}

*Gene Editing
*CRISPR-Cas Systems

@article {pmid38564197,
year = {2024},
author = {Lima, L and Berni, M and Mota, J and Bressan, D and Julio, A and Cavalcante, R and Macias, V and Li, Z and Rasgon, JL and Bier, E and Araujo, H},
title = {Gene Editing in the Chagas Disease Vector Rhodnius prolixus by Cas9-Mediated ReMOT Control.},
journal = {The CRISPR journal},
volume = {7},
number = {2},
pages = {88-99},
doi = {10.1089/crispr.2023.0076},
pmid = {38564197},
issn = {2573-1602},
mesh = {Animals ; Female ; Gene Editing/methods ; *Rhodnius/genetics/parasitology ; CRISPR-Cas Systems ; Insect Vectors/parasitology ; *Chagas Disease/genetics/parasitology ; },
abstract = {Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease.},
}

Animals
Female
Gene Editing/methods
*Rhodnius/genetics/parasitology
CRISPR-Cas Systems
Insect Vectors/parasitology
*Chagas Disease/genetics/parasitology

@article {pmid38447647,
year = {2024},
author = {Wang, W and Wang, S and Meng, X and Zhao, Y and Li, N and Wang, T and Feng, N and Yan, F and Xia, X},
title = {A virus-like particle candidate vaccine based on CRISPR/Cas9 gene editing technology elicits broad-spectrum protection against SARS-CoV-2.},
journal = {Antiviral research},
volume = {225},
number = {},
pages = {105854},
doi = {10.1016/j.antiviral.2024.105854},
pmid = {38447647},
issn = {1872-9096},
mesh = {Mice ; Animals ; Humans ; SARS-CoV-2 ; *Vaccines, Virus-Like Particle ; COVID-19 Vaccines ; CRISPR-Cas Systems ; Gene Editing ; *COVID-19 ; Antibodies, Viral ; Antibodies, Neutralizing ; },
abstract = {The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with frequent mutations has seriously damaged the effectiveness of the 2019 coronavirus disease (COVID-19) vaccine. There is an urgent need to develop a broad-spectrum vaccine while elucidating the underlying immune mechanisms. Here, we developed a SARS-CoV-2 virus-like particles (VLPs) vaccine based on the Canarypox-virus vector (ALVAC-VLPs) using CRISPR/Cas9. Immunization with ALVAC-VLPs showed the effectively induce SARS-CoV-2 specific T and B cell responses to resist the lethal challenge of mouse adaptive strains. Notably, ALVAC-VLPs conferred protection in golden hamsters against SARS-CoV-2 Wuhan-Hu-1 (wild-type, WT) and variants (Beta, Delta, Omicron BA.1, and BA.2), as evidenced by the prevention of weight loss, reduction in lung and turbinate tissue damage, and decreased viral load. Further investigation into the mechanism of immune response induced by ALVAC-VLPs revealed that toll-like receptor 4 (TLR4) mediates the recruitment of dendritic cells (DCs) to secondary lymphoid organs, thereby initiating follicle assisted T (Tfh) cell differentiation, the proliferation of germinal center (GC) B cells and plasma cell production. These findings demonstrate the immunogenicity and efficacy of the safe ALVAC-VLPs vaccine against SARS-CoV-2 and provide valuable insight into the development of COVID-19 vaccine strategies.},
}

Mice
Animals
Humans
SARS-CoV-2
*Vaccines, Virus-Like Particle
COVID-19 Vaccines
CRISPR-Cas Systems
Gene Editing
*COVID-19
Antibodies, Viral
Antibodies, Neutralizing

@article {pmid38366553,
year = {2024},
author = {Zhang, T and Zhao, F and Li, J and Sun, X and Zhang, X and Wang, H and Fan, P and Lai, L and Li, Z and Sui, T},
title = {Programmable RNA 5-methylcytosine (m5C) modification of cellular RNAs by dCasRx conjugated methyltransferase and demethylase.},
journal = {Nucleic acids research},
volume = {52},
number = {6},
pages = {2776-2791},
pmid = {38366553},
issn = {1362-4962},
support = {32101226//National Natural Science Foundation of China/ ; YESS20210189//China Association for Science and Technology/ ; },
mesh = {Animals ; Mice ; *5-Methylcytosine/metabolism ; *Methylation ; *Methyltransferases/genetics/metabolism ; RNA, Transfer/metabolism ; *Genetic Techniques ; CRISPR-Cas Systems ; Humans ; *RNA Editing ; },
abstract = {5-Methylcytosine (m5C), an abundant RNA modification, plays a crucial role in regulating RNA fate and gene expression. While recent progress has been made in understanding the biological roles of m5C, the inability to introduce m5C at specific sites within transcripts has hindered efforts to elucidate direct links between specific m5C and phenotypic outcomes. Here, we developed a CRISPR-Cas13d-based tool, named reengineered m5C modification system (termed 'RCMS'), for targeted m5C methylation and demethylation in specific transcripts. The RCMS editors consist of a nuclear-localized dCasRx conjugated to either a methyltransferase, NSUN2/NSUN6, or a demethylase, the catalytic domain of mouse Tet2 (ten-eleven translocation 2), enabling the manipulation of methylation events at precise m5C sites. We demonstrate that the RCMS editors can direct site-specific m5C incorporation and demethylation. Furthermore, we confirm their effectiveness in modulating m5C levels within transfer RNAs and their ability to induce changes in transcript abundance and cell proliferation through m5C-mediated mechanisms. These findings collectively establish RCMS editors as a focused epitranscriptome engineering tool, facilitating the identification of individual m5C alterations and their consequential effects.},
}

Animals
Mice
*5-Methylcytosine/metabolism
*Methylation
*Methyltransferases/genetics/metabolism
RNA, Transfer/metabolism
*Genetic Techniques
CRISPR-Cas Systems
Humans
*RNA Editing

@article {pmid38149939,
year = {2024},
author = {Guri-Lamce, I and AlRokh, Y and Kim, Y and Maeshima, R and Graham, C and Hart, SL and McGrath, JA and Jacków-Malinowska, J},
title = {Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases?.},
journal = {The British journal of dermatology},
volume = {190},
number = {5},
pages = {617-627},
doi = {10.1093/bjd/ljad528},
pmid = {38149939},
issn = {1365-2133},
mesh = {Humans ; Gene Editing ; CRISPR-Cas Systems ; Gene Transfer Techniques ; *Nanoparticles ; *Skin Diseases, Genetic/genetics ; *Liposomes ; },
abstract = {Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.},
}

Humans
Gene Editing
CRISPR-Cas Systems
Gene Transfer Techniques
*Nanoparticles
*Skin Diseases, Genetic/genetics
*Liposomes

@article {pmid38609262,
year = {2024},
author = {Su, Z and Wang, X and Chen, X and Ding, L and Zeng, X and Xu, J and Peng, C},
title = {Novel CRISPR/SpRY system for rapid detection of CRISPR/Cas-mediated gene editing in rice.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342519},
doi = {10.1016/j.aca.2024.342519},
pmid = {38609262},
issn = {1873-4324},
mesh = {*Oryza/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; Biological Assay ; Biotechnology ; RNA ; },
abstract = {The gene editing technology represented by clustered rule-interspersed short palindromic repeats (CRISPR)/Cas9 has developed as a common tool in the field of biotechnology. Many gene-edited products in plant varieties have recently been commercialized. However, the rapid on-site visual detection of gene-edited products without instrumentation remains challenging. This study aimed to develop a novel and efficient method, termed the CRISPR/SpRY detection platform, for the rapid screening of CRISPR/Cas9-induced mutants based on CRISPR/SpRY-mediated in vitro cleavage using rice (Oryza sativa L.) samples genetically edited at the TGW locus as an example. We designed the workflow of the CRISPR/SpRY detection platform and conducted a feasibility assessment. Subsequently, we optimized the reaction system of CRISPR/SpRY, and developed a one-pot CRISPR/SpRY assay by integrating recombinase polymerase amplification (RPA). The sensitivity of the method was further verified using recombinant plasmids. The proposed method successfully identified various types of mutations, including insertions, deletions (indels), and nucleotide substitutions, with excellent sensitivity. Finally, the applicability of this method was validated using different rice samples. The entire process was completed in less than an hour, with a limit of detection as low as 1%. Compared with previous methods, our approach is simple to operate, instrumentation-free, cost-effective, and time-efficient. The primary significance lies in the liberation of our developed system from the limitations imposed using protospacer adjacent motif sequences. This expands the scope and versatility of the CRISPR-based detection platform, making it a promising and groundbreaking platform for detecting mutations induced by gene editing.},
}

*Oryza/genetics
CRISPR-Cas Systems/genetics
Gene Editing
Biological Assay
Biotechnology
RNA

@article {pmid38609257,
year = {2024},
author = {He, W and Li, X and Li, X and Guo, M and Zhang, M and Hu, R and Li, M and Ding, S and Yan, Y},
title = {Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1303},
number = {},
pages = {342477},
doi = {10.1016/j.aca.2024.342477},
pmid = {38609257},
issn = {1873-4324},
mesh = {*MicroRNAs/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; *Nucleic Acids ; DNA, Single-Stranded/genetics ; },
abstract = {CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.},
}

*MicroRNAs/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
*Nucleic Acids
DNA, Single-Stranded/genetics

@article {pmid38609223,
year = {2024},
author = {Ma, J and Qian, C and Hu, Q and Zhang, J and Gu, G and Liang, X and Zhang, L},
title = {The bacteriome-coupled phage communities continuously contract and shift to orchestrate the traditional rice vinegar fermentation.},
journal = {Food research international (Ottawa, Ont.)},
volume = {184},
number = {},
pages = {114244},
doi = {10.1016/j.foodres.2024.114244},
pmid = {38609223},
issn = {1873-7145},
mesh = {Humans ; *Oryza ; Acetic Acid ; Fermentation ; *Bacteriophages/genetics ; *Microbiota/genetics ; },
abstract = {Amounts of microbiome studies have uncovered the microbial communities of traditional food fermentations, while in which the phageome development with time is poorly understood. Here, we conducted a study to decipher both phageome and bacteriome of the traditional rice vinegar fermentation. The vinegar phageomes showed significant differences in the alpha diversity, network density and clustering coefficient over time. Peduoviridae had the highest relative abundance. Moreover, the phageome negatively correlated to the cognate bacteriome in alpha diversity, and undergone constantly contracting and shifting across the temporal scale. Nevertheless, 257 core virial clusters (VCs) persistently occurred with time whatever the significant impacts imposed by the varied physiochemical properties. Glycoside hydrolase (GH) and glycosyltransferase (GT) families genes displayed the higher abundances across all samples. Intriguingly, diversely structuring of toxin-antitoxin systems (TAs) and CRISPR-Cas arrays were frequently harbored by phage genomes. Their divergent organization and encoding attributes underlie the multiple biological roles in modulation of network and/or contest of phage community as well as bacterial host community. This phageome-wide mapping will fuel the current insights of phage community ecology in other traditional fermented ecosystems that are challenging to decipher.},
}

Humans
*Oryza
Acetic Acid
Fermentation
*Bacteriophages/genetics
*Microbiota/genetics

@article {pmid38607027,
year = {2024},
author = {Chua, R and Wang, L and Singaraja, R and Ghosh, S},
title = {Functional and Multi-Omics Effects of an Optimized CRISPR-Mediated FURIN Depletion in U937 Monocytes.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/cells13070588},
pmid = {38607027},
issn = {2073-4409},
support = {2U54GM104940, R01HL146462-01/NH/NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; *Monocytes/metabolism ; *Furin/genetics/metabolism ; U937 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; Multiomics ; RNA, Guide, CRISPR-Cas Systems ; Cytokines/genetics ; },
abstract = {The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.},
}

Humans
Animals
Mice
*Monocytes/metabolism
*Furin/genetics/metabolism
U937 Cells
Clustered Regularly Interspaced Short Palindromic Repeats
Multiomics
RNA, Guide, CRISPR-Cas Systems
Cytokines/genetics

@article {pmid38607007,
year = {2024},
author = {Wang, S and Zhu, Y and Du, S and Zheng, Y},
title = {Preclinical Advances in LNP-CRISPR Therapeutics for Solid Tumor Treatment.},
journal = {Cells},
volume = {13},
number = {7},
pages = {},
pmid = {38607007},
issn = {2073-4409},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; Gene Editing ; Genetic Therapy ; *Neoplasms/genetics/therapy ; *Nanostructures ; },
abstract = {Solid tumors, with their intricate cellular architecture and genetic heterogeneity, have long posed therapeutic challenges. The advent of the CRISPR genome editing system offers a promising, precise genetic intervention. However, the journey from bench to bedside is fraught with hurdles, chief among them being the efficient delivery of CRISPR components to tumor cells. Lipid nanoparticles (LNPs) have emerged as a potential solution. This biocompatible nanomaterial can encapsulate the CRISPR/Cas9 system, ensuring targeted delivery while mitigating off-target effects. Pre-clinical investigations underscore the efficacy of LNP-mediated CRISPR delivery, with marked disruption of oncogenic pathways and subsequent tumor regression. Overall, CRISPR/Cas9 technology, when combined with LNPs, presents a groundbreaking approach to cancer therapy, offering precision, efficacy, and potential solutions to current limitations. While further research and clinical testing are required, the future of personalized cancer treatment based on CRISPR/Cas9 holds immense promise.},
}

Humans
CRISPR-Cas Systems/genetics
Gene Editing
Genetic Therapy
*Neoplasms/genetics/therapy
*Nanostructures

@article {pmid38605715,
year = {2024},
author = {Shen, Q and Ruan, H and Zhang, H and Wu, T and Zhu, K and Han, W and Dong, R and Ming, T and Qi, H and Zhang, Y},
title = {Utilization of CRISPR-Cas genome editing technology in filamentous fungi: function and advancement potentiality.},
journal = {Frontiers in microbiology},
volume = {15},
number = {},
pages = {1375120},
pmid = {38605715},
issn = {1664-302X},
abstract = {Filamentous fungi play a crucial role in environmental pollution control, protein secretion, and the production of active secondary metabolites. The evolution of gene editing technology has significantly improved the study of filamentous fungi, which in the past was laborious and time-consuming. But recently, CRISPR-Cas systems, which utilize small guide RNA (sgRNA) to mediate clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), have demonstrated considerable promise in research and application for filamentous fungi. The principle, function, and classification of CRISPR-Cas, along with its application strategies and research progress in filamentous fungi, will all be covered in the review. Additionally, we will go over general matters to take into account when editing a genome with the CRISPR-Cas system, including the creation of vectors, different transformation methodologies, multiple editing approaches, CRISPR-mediated transcriptional activation (CRISPRa) or interference (CRISPRi), base editors (BEs), and Prime editors (PEs).},
}

@article {pmid38578074,
year = {2024},
author = {Meng, JN and Xu, ZK and Li, PR and Zeng, X and Liu, Y and Xu, ZL and Wang, J and Ding, Y and Shen, X},
title = {Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8823-8830},
doi = {10.1021/acs.jafc.3c06744},
pmid = {38578074},
issn = {1520-5118},
mesh = {*Food Microbiology ; *Emetics ; Recombinases/genetics ; Bacillus cereus/genetics ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Nucleotidyltransferases/genetics ; },
abstract = {Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10[-2] ng μL[-1] and 10[2] CFU mL[-1], respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.},
}

*Food Microbiology
*Emetics
Recombinases/genetics
Bacillus cereus/genetics
CRISPR-Cas Systems
Sensitivity and Specificity
Nucleotidyltransferases/genetics

@article {pmid38575365,
year = {2024},
author = {Wang, Y and Du, P and Shao, Y and Wang, W and Liu, Y and Ma, Y and Hu, P and Cao, J and Wang, X and Abd El-Aty, AM},
title = {An Innovative and Efficient Fluorescent Detection Technique for Salmonella in Animal-Derived Foods Using the CRISPR/Cas12a-HCR System Combined with PCR/RAA.},
journal = {Journal of agricultural and food chemistry},
volume = {72},
number = {15},
pages = {8831-8839},
doi = {10.1021/acs.jafc.3c08829},
pmid = {38575365},
issn = {1520-5118},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; Coloring Agents ; DNA, Single-Stranded ; Recombinases ; Salmonella/genetics ; Polymerase Chain Reaction ; *Biosensing Techniques ; },
abstract = {Here, we present a method for Salmonella detection using clustered regularly interspaced short palindromic repeats associated with the CRISPR-associated protein 12a-hybridization chain reaction (CRISPR/Cas12a-HCR) system combined with polymerase chain reaction/recombinase-assisted amplification (PCR/RAA) technology. The approach relies on the Salmonella invA gene as a biorecognition element and its amplification through PCR and RAA. In the presence of the target gene, Cas12a, guided by crRNA, recognizes and cleaves the amplification product, initiating the HCR. Fluorescently labeled single-stranded DNA (ssDNA) H1 and H2 were introduced, and the Salmonella concentration was determined based on the fluorescence intensity from the triggered HCR. Both assays demonstrate high specificity, sensitivity, simplicity, and rapidity. The detection range was 2 × 10[1]-2 × 10[9] CFU/mL, with an LOD of 20 CFU/mL, and the entire process enabled specific and rapid Salmonella detection within 85-105 min. Field-incurred spiked recovery tests were conducted in mutton and beef samples using both assays, demonstrating satisfactory recovery and accuracy in animal-derived foods. By combining CRISPR/Cas12a with hybridization chain reaction technology, this study presents a rapid and sensitive Salmonella detection method that is crucial for identifying pathogenic bacteria and monitoring food safety.},
}

Animals
Cattle
*CRISPR-Cas Systems
Coloring Agents
DNA, Single-Stranded
Recombinases
Salmonella/genetics
Polymerase Chain Reaction
*Biosensing Techniques

@article {pmid38564120,
year = {2024},
author = {Sarkar, P and Santiago Vazquez, J and Zhou, M and Levy, A and Mou, Z and Orbović, V},
title = {Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.},
journal = {Transgenic research},
volume = {33},
number = {1-2},
pages = {59-66},
pmid = {38564120},
issn = {1573-9368},
support = {2018-70016-27392//USDA/NIFA Emergency Citrus Disease Research and Extension Program/ ; },
mesh = {*Gene Editing ; CRISPR-Cas Systems/genetics ; Introns ; *Citrus/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Transfer/genetics ; },
abstract = {Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.},
}

*Gene Editing
CRISPR-Cas Systems/genetics
Introns
*Citrus/genetics
RNA, Guide, CRISPR-Cas Systems
RNA, Transfer/genetics

@article {pmid38504013,
year = {2024},
author = {Abkallo, HM and Arbuthnot, P and Auer, TO and Berger, DK and Burger, J and Chakauya, E and Concordet, JP and Diabate, A and Di Donato, V and Groenewald, JH and Guindo, A and Koekemoer, LL and Nazare, F and Nolan, T and Okumu, F and Orefuwa, E and Paemka, L and Prieto-Godino, L and Runo, S and Sadler, M and Tesfaye, K and Tripathi, L and Wondji, C},
title = {Making genome editing a success story in Africa.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {551-554},
pmid = {38504013},
issn = {1546-1696},
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; Africa ; Genome ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics
Africa
Genome

@article {pmid38497640,
year = {2024},
author = {Wang, P and Du, X and Zhao, Y and Wang, W and Cai, T and Tang, K and Wang, X},
title = {Combining CRISPR/Cas9 and natural excision for the precise and complete removal of mobile genetic elements in bacteria.},
journal = {Applied and environmental microbiology},
volume = {90},
number = {4},
pages = {e0009524},
pmid = {38497640},
issn = {1098-5336},
support = {32070175//MOST | National Natural Science Foundation of China (NSFC)/ ; 42188102//MOST | National Natural Science Foundation of China (NSFC)/ ; 91951203//MOST | National Natural Science Foundation of China (NSFC)/ ; 2022YFC3103600//MOST | National Key Research and Development Program of China (NKPs)/ ; 2019BT02Y262//Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program/ ; 2021345//Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS YIPA)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems ; Bacteria/genetics ; Genomic Islands ; Gene Transfer, Horizontal ; Plasmids/genetics ; Interspersed Repetitive Sequences ; },
abstract = {Horizontal gene transfer, facilitated by mobile genetic elements (MGEs), is an adaptive evolutionary process that contributes to the evolution of bacterial populations and infectious diseases. A variety of MGEs not only can integrate into the bacterial genome but also can survive or even replicate like plasmids in the cytoplasm, thus requiring precise and complete removal for studying their strategies in benefiting host cells. Existing methods for MGE removal, such as homologous recombination-based deletion and excisionase-based methods, have limitations in effectively eliminating certain MGEs. To overcome these limitations, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision of MGEs. In this approach, a specialized single guide RNA (sgRNA) element is designed with a 20-nucleotide region that pairs with the MGE sequence. This sgRNA is expressed from a plasmid that also carries the Cas9 gene. By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be precisely and completely eliminated through Cas9 cleavage, generating MGE-removed cells. We have successfully applied the Cas9-NE method to remove four representative MGEs, including plasmids, prophages, and genomic islands, from Vibrio strains. This new approach not only enables various investigations on MGEs but also has significant implications for the rapid generation of strains for commercial purposes.IMPORTANCEMobile genetic elements (MGEs) are of utmost importance for bacterial adaptation and pathogenicity, existing in various forms and multiple copies within bacterial cells. Integrated MGEs play dual roles in bacterial hosts, enhancing the fitness of the host by delivering cargo genes and potentially modifying the bacterial genome through the integration/excision process. This process can lead to alterations in promoters or coding sequences or even gene disruptions at integration sites, influencing the physiological functions of host bacteria. Here, we developed a new approach called Cas9-NE, allowing them to maintain the natural sequence changes associated with MGE excision. Cas9-NE allows the one-step removal of integrated and circular MGEs, addressing the challenge of eliminating various MGE forms efficiently. This approach simplifies MGE elimination in bacteria, expediting research on MGEs.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems
Bacteria/genetics
Genomic Islands
Gene Transfer, Horizontal
Plasmids/genetics
Interspersed Repetitive Sequences

@article {pmid38323580,
year = {2024},
author = {Romano, A and Mortellaro, A},
title = {The New Frontiers of Gene Therapy and Gene Editing in Inflammatory Diseases.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {219-231},
doi = {10.1089/hum.2023.210},
pmid = {38323580},
issn = {1557-7422},
mesh = {Animals ; Humans ; *Gene Editing/methods ; Quality of Life ; Genetic Therapy/methods ; Genetic Engineering ; *Severe Combined Immunodeficiency ; CRISPR-Cas Systems ; },
abstract = {Inflammatory diseases are conditions characterized by abnormal and often excessive immune responses, leading to tissue and organ inflammation. The complexity of these disorders arises from the intricate interplay of genetic factors and immune responses, which challenges conventional therapeutic approaches. However, the field of genetic manipulation has sparked unprecedented optimism in addressing these complex disorders. This review aims to comprehensively explore the application of gene therapy and gene editing in the context of inflammatory diseases, offering solutions that range from correcting genetic defects to precise immune modulation. These therapies have exhibited remarkable potential in ameliorating symptoms, improving quality of life, and even achieving disease remission. As we delve into recent breakthroughs and therapeutic applications, we illustrate how these advancements offer novel and transformative solutions for conditions that have traditionally eluded conventional treatments. By examining successful case studies and preclinical research, we emphasize the favorable results and substantial transformative impacts that gene-based interventions have demonstrated in patients and animal models of inflammatory diseases such as chronic granulomatous disease, cryopyrin-associated syndromes, and adenosine deaminase 2 deficiency, as well as those of multifactorial origins such as arthropathies (osteoarthritis, rheumatoid arthritis) and inflammatory bowel disease. In conclusion, gene therapy and gene editing offer transformative opportunities to address the underlying causes of inflammatory diseases, ushering in a new era of precision medicine and providing hope for personalized, targeted treatments.},
}

Animals
Humans
*Gene Editing/methods
Quality of Life
Genetic Therapy/methods
Genetic Engineering
*Severe Combined Immunodeficiency
CRISPR-Cas Systems

@article {pmid38251667,
year = {2024},
author = {Rai, R and Steinberg, Z and Romito, M and Zinghirino, F and Hu, YT and White, N and Naseem, A and Thrasher, AJ and Turchiano, G and Cavazza, A},
title = {CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {269-283},
doi = {10.1089/hum.2023.100},
pmid = {38251667},
issn = {1557-7422},
mesh = {Infant, Newborn ; Humans ; *Severe Combined Immunodeficiency/genetics/therapy ; T-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Gene Editing/methods ; Receptors, Interleukin-7/genetics/metabolism ; },
abstract = {Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.},
}

Infant, Newborn
Humans
*Severe Combined Immunodeficiency/genetics/therapy
T-Lymphocytes/metabolism
CRISPR-Cas Systems
Hematopoietic Stem Cells/metabolism
Gene Editing/methods
Receptors, Interleukin-7/genetics/metabolism

@article {pmid38159103,
year = {2024},
author = {Wang, R and Li, Y and Xu, S and Huang, Q and Tu, M and Zhu, Y and Cen, H and Dong, J and Jiang, L and Yao, X},
title = {Genome-wide association study reveals the genetic basis for petal-size formation in rapeseed (Brassica napus) and CRISPR-Cas9-mediated mutagenesis of BnFHY3 for petal-size reduction.},
journal = {The Plant journal : for cell and molecular biology},
volume = {118},
number = {2},
pages = {373-387},
doi = {10.1111/tpj.16609},
pmid = {38159103},
issn = {1365-313X},
support = {2022YFD1200404//National Key Research and Development Program of China/ ; },
mesh = {*Brassica napus/genetics ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Plant Breeding ; *Brassica rapa/genetics ; Mutagenesis ; },
abstract = {Petals in rapeseed (Brassica napus) serve multiple functions, including protection of reproductive organs, nutrient acquisition, and attraction of pollinators. However, they also cluster densely at the top, forming a thick layer that absorbs and reflects a considerable amount of photosynthetically active radiation. Breeding genotypes with large, small, or even petal-less varieties, requires knowledge of primary genes for allelic selection and manipulation. However, our current understanding of petal-size regulation is limited, and the lack of markers and pre-breeding materials hinders targeted petal-size breeding. Here, we conducted a genome-wide association study on petal size using 295 diverse accessions. We identified 20 significant single nucleotide polymorphisms and 236 genes associated with petal-size variation. Through a cross-analysis of genomic and transcriptomic data, we focused on 14 specific genes, from which molecular markers for diverging petal-size features can be developed. Leveraging CRISPR-Cas9 technology, we successfully generated a quadruple mutant of Far-Red Elongated Hypocotyl 3 (q-bnfhy3), which exhibited smaller petals compared to the wild type. Our study provides insights into the genetic basis of petal-size regulation in rapeseed and offers abundant potential molecular markers for breeding. The q-bnfhy3 mutant unveiled a novel role of FHY3 orthologues in regulating petal size in addition to previously reported functions.},
}

*Brassica napus/genetics
Genome-Wide Association Study
CRISPR-Cas Systems
Plant Breeding
*Brassica rapa/genetics
Mutagenesis

@article {pmid38069547,
year = {2024},
author = {Zhang, M and Feng, J and Li, Y and Qin, PZ and Chai, Y},
title = {Generation of tamoxifen-inducible Tfap2b-CreER[T2] mice using CRISPR-Cas9.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {62},
number = {1},
pages = {e23582},
pmid = {38069547},
issn = {1526-968X},
support = {R01 DE012711/NH/NIH HHS/United States ; R01 DE022503/NH/NIH HHS/United States ; R01 DE012711/DE/NIDCR NIH HHS/United States ; T90 DE021982/DE/NIDCR NIH HHS/United States ; R01 DE022503/DE/NIDCR NIH HHS/United States ; },
mesh = {Mice ; Animals ; *CRISPR-Cas Systems ; *Tamoxifen/pharmacology ; Mice, Transgenic ; Red Fluorescent Protein ; Integrases/genetics/metabolism ; },
abstract = {Tfap2b, a pivotal transcription factor, plays critical roles within neural crest cells and their derived lineage. To unravel the intricate lineage dynamics and contribution of these Tfap2b+ cells during craniofacial development, we established a Tfap2b-CreER[T2] knock-in transgenic mouse line using the CRISPR-Cas9-mediated homologous direct repair. By breeding with tdTomato reporter mice and initiating Cre activity through tamoxifen induction at distinct developmental time points, we show the Tfap2b lineage within the key neural crest-derived domains, such as the facial mesenchyme, midbrain, cerebellum, spinal cord, and limbs. Notably, the migratory neurons stemming from the dorsal root ganglia are visible subsequent to Cre activity initiated at E8.5. Intriguingly, Tfap2b+ cells, serving as the progenitors for limb development, show activity predominantly commencing at E10.5. Across the mouse craniofacial landscape, Tfap2b exhibits a widespread presence throughout the facial organs. Here we validate its role as a marker of progenitors in tooth development and have confirmed that this process initiates from E12.5. Our study not only validates the Tfap2b-CreER[T2] transgenic line, but also provides a powerful tool for lineage tracing and genetic targeting of Tfap2b-expressing cells and their progenitor in a temporally and spatially regulated manner during the intricate process of development and organogenesis.},
}

Mice
Animals
*CRISPR-Cas Systems
*Tamoxifen/pharmacology
Mice, Transgenic
Red Fluorescent Protein
Integrases/genetics/metabolism

@article {pmid38062734,
year = {2024},
author = {Whittaker, TE and Moula, SE and Bahal, S and Bakri, FG and Hayajneh, WA and Daoud, AK and Naseem, A and Cavazza, A and Thrasher, AJ and Santilli, G},
title = {Multidimensional Response Surface Methodology for the Development of a Gene Editing Protocol for p67[phox]-Deficient Chronic Granulomatous Disease.},
journal = {Human gene therapy},
volume = {35},
number = {7-8},
pages = {298-312},
pmid = {38062734},
issn = {1557-7422},
support = {/WT_/Wellcome Trust/United Kingdom ; 104807/WT_/Wellcome Trust/United Kingdom ; MR/S021930/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Granulomatous Disease, Chronic/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; Antigens, CD34/genetics ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Replacing a faulty gene with a correct copy has become a viable therapeutic option as a result of recent progress in gene editing protocols. Targeted integration of therapeutic genes in hematopoietic stem cells has been achieved for multiple genes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system and Adeno-Associated Virus (AAV) to carry a donor template. Although this is a promising strategy to correct genetic blood disorders, it is associated with toxicity and loss of function in CD34[+] hematopoietic stem and progenitor cells, which has hampered clinical application. Balancing the maximum achievable correction against deleterious effects on the cells is critical. However, multiple factors are known to contribute, and the optimization process is laborious and not always clearly defined. We have developed a flexible multidimensional Response Surface Methodology approach for optimization of gene correction. Using this approach, we could rapidly investigate and select editing conditions for CD34[+] cells with the best possible balance between correction and cell/colony-forming unit (CFU) loss in a parsimonious one-shot experiment. This method revealed that using relatively low doses of AAV2/6 and CRISPR/Cas9 ribonucleoprotein complex, we can preserve the fitness of CD34[+] cells and, at the same time, achieve high levels of targeted gene insertion. We then used these optimized editing conditions for the correction of p67[phox]-deficient chronic granulomatous disease (CGD), an autosomal recessive disorder of blood phagocytic cells resulting in severe recurrent bacterial and fungal infections and achieved rescue of p67[phox] expression and functional correction of CD34[+]-derived neutrophils from a CGD patient.},
}

Humans
*Granulomatous Disease, Chronic/genetics/therapy
Gene Editing
Genetic Therapy/methods
Antigens, CD34/genetics
Hematopoietic Stem Cells/metabolism
CRISPR-Cas Systems

@article {pmid37997697,
year = {2024},
author = {Zhang, D and Boch, J},
title = {Development of TALE-adenine base editors in plants.},
journal = {Plant biotechnology journal},
volume = {22},
number = {5},
pages = {1067-1077},
pmid = {37997697},
issn = {1467-7652},
mesh = {Humans ; *Cytosine Deaminase/genetics ; *Adenine ; Gene Editing/methods ; DNA/genetics ; CRISPR-Cas Systems ; *Cytidine Deaminase ; *Proteins ; },
abstract = {Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a β-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.},
}

Humans
*Cytosine Deaminase/genetics
*Adenine
Gene Editing/methods
DNA/genetics
CRISPR-Cas Systems
*Cytidine Deaminase
*Proteins

@article {pmid37400521,
year = {2024},
author = {Wessels, HH and Stirn, A and Méndez-Mancilla, A and Kim, EJ and Hart, SK and Knowles, DA and Sanjana, NE},
title = {Prediction of on-target and off-target activity of CRISPR-Cas13d guide RNAs using deep learning.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {628-637},
pmid = {37400521},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Deep Learning ; RNA ; Gene Editing ; },
abstract = {Transcriptome engineering applications in living cells with RNA-targeting CRISPR effectors depend on accurate prediction of on-target activity and off-target avoidance. Here we design and test ~200,000 RfxCas13d guide RNAs targeting essential genes in human cells with systematically designed mismatches and insertions and deletions (indels). We find that mismatches and indels have a position- and context-dependent impact on Cas13d activity, and mismatches that result in G-U wobble pairings are better tolerated than other single-base mismatches. Using this large-scale dataset, we train a convolutional neural network that we term targeted inhibition of gene expression via gRNA design (TIGER) to predict efficacy from guide sequence and context. TIGER outperforms the existing models at predicting on-target and off-target activity on our dataset and published datasets. We show that TIGER scoring combined with specific mismatches yields the first general framework to modulate transcript expression, enabling the use of RNA-targeting CRISPRs to precisely control gene dosage.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
*Deep Learning
RNA
Gene Editing

@article {pmid37365261,
year = {2024},
author = {},
title = {Efficient A-to-C base editing with high specificity.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {578-579},
pmid = {37365261},
issn = {1546-1696},
support = {32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing ; *CRISPR-Cas Systems/genetics ; },
}

*Gene Editing
*CRISPR-Cas Systems/genetics

@article {pmid37322276,
year = {2024},
author = {Chen, L and Hong, M and Luan, C and Gao, H and Ru, G and Guo, X and Zhang, D and Zhang, S and Li, C and Wu, J and Randolph, PB and Sousa, AA and Qu, C and Zhu, Y and Guan, Y and Wang, L and Liu, M and Feng, B and Song, G and Liu, DR and Li, D},
title = {Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.},
journal = {Nature biotechnology},
volume = {42},
number = {4},
pages = {638-650},
pmid = {37322276},
issn = {1546-1696},
support = {82100773//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; U01AI142756//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing ; *Adenine/metabolism ; Mutation ; Cytosine/metabolism ; Adenosine ; CRISPR-Cas Systems/genetics ; Mammals/genetics ; },
abstract = {Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.},
}

Animals
Mice
Humans
*Gene Editing
*Adenine/metabolism
Mutation
Cytosine/metabolism
Adenosine
CRISPR-Cas Systems/genetics
Mammals/genetics

@article {pmid38605260,
year = {2024},
author = {Rafiq, MS and Shabbir, MA and Raza, A and Irshad, S and Asghar, A and Maan, MK and Gondal, MA and Hao, H},
title = {CRISPR-Cas System: A New Dawn to Combat Antibiotic Resistance.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
pmid = {38605260},
issn = {1179-190X},
support = {32172914//the National Natural Science Foundation of China/ ; 2021YFD1800600//Key Technology Research and Development Program of Shandong Province/ ; . 2662022DKYJC005//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Antimicrobial resistance (AMR) can potentially harm global public health. Horizontal gene transfer (HGT), which speeds up the emergence of AMR and increases the burden of drug resistance in mobile genetic elements (MGEs), is the primary method by which AMR genes are transferred across bacterial pathogens. New approaches are urgently needed to halt the spread of bacterial diseases and antibiotic resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an RNA-guided adaptive immune system, protects prokaryotes from foreign DNA like plasmids and phages. This approach may be essential in limiting horizontal gene transfer and halting the spread of antibiotic resistance. The CRISPR-Cas system has been crucial in identifying and understanding resistance mechanisms and developing novel therapeutic approaches. This review article investigates the CRISPR-Cas system's potential as a tool to combat bacterial AMR. Antibiotic-resistant bacteria can be targeted and eliminated by the CRISPR-Cas system. It has been proven to be an efficient method for removing carbapenem-resistant plasmids and regaining antibiotic susceptibility. The CRISPR-Cas system has enormous potential as a weapon against bacterial AMR. It precisely targets and eliminates antibiotic-resistant bacteria, facilitates resistance mechanism identification, and offers new possibilities in diagnostics and therapeutics.},
}

@article {pmid38605095,
year = {2024},
author = {de Boer, EN and Vroom, V and Scheper, AJ and Johansson, LF and Bosscher, L and Rietema, N and Commandeur-Jan, SZ and Knoers, NVAM and Sikkema-Raddatz, B and van den Berg, E and van Diemen, CC},
title = {Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8508},
pmid = {38605095},
issn = {2045-2322},
mesh = {Humans ; Reproducibility of Results ; *CRISPR-Cas Systems ; Karyotyping ; *Leukemia ; Chromosome Mapping ; },
abstract = {Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.},
}

Humans
Reproducibility of Results
*CRISPR-Cas Systems
Karyotyping
*Leukemia
Chromosome Mapping

@article {pmid38603815,
year = {2024},
author = {Bai, H and He, LY and Gao, FZ and Yao, KS and Zhang, M and Qiao, LK and Chen, ZY and He, LX and Liu, YS and Zhao, JL and Ying, GG},
title = {Airborne antibiotic resistome and microbiome in pharmaceutical factories.},
journal = {Environment international},
volume = {186},
number = {},
pages = {108639},
doi = {10.1016/j.envint.2024.108639},
pmid = {38603815},
issn = {1873-6750},
abstract = {Antimicrobial resistance is considered to be one of the biggest public health problems, and airborne transmission is an important but under-appreciated pathway for the spread of antibiotic resistance genes (ARGs) in the environment. Previous research has shown pharmaceutical factories to be a major source of ARGs and antibiotic resistant bacteria (ARB) in the surrounding receiving water and soil environments. Pharmaceutical factories are hotspots of antibiotic resistance, but the atmospheric transmission and its environmental risk remain more concerns. Here, we conducted a metagenomic investigation into the airborne microbiome and resistome in three pharmaceutical factories in China. Soil (average: 38.45%) and wastewater (average: 28.53%) were major contributors of airborne resistome. ARGs (vanR/vanS, blaOXA, and CfxA) conferring resistance to critically important clinically used antibiotics were identified in the air samples. The wastewater treatment area had significantly higher relative abundances of ARGs (average: 0.64 copies/16S rRNA). Approximately 28.2% of the detected airborne ARGs were found to be associated with plasmids, and this increased to about 50% in the wastewater treatment area. We have compiled a list of high-risk airborne ARGs found in pharmaceutical factories. Moreover, A total of 1,043 viral operational taxonomic units were identified and linked to 47 family-group taxa. Different CRISPR-Cas immune systems have been identified in bacterial hosts in response to phage infection. Similarly, higher phage abundance (average: 2451.70 PPM) was found in the air of the wastewater treatment area. Our data provide insights into the antibiotic resistance gene profiles and microbiome (bacterial and non-bacterial) in pharmaceutical factories and reveal the potential role of horizontal transfer in the spread of airborne ARGs, with implications for human and animal health.},
}

@article {pmid38602884,
year = {2024},
author = {van Beljouw, SPB and Haagsma, AC and Kalogeropoulos, K and Pabst, M and Brouns, SJJ},
title = {Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.3c00788},
pmid = {38602884},
issn = {1554-8937},
abstract = {The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.},
}

@article {pmid38600465,
year = {2024},
author = {Ci, Y and Zhang, Y and Zhang, X},
title = {Methylated lncRNAs suppress apoptosis of gastric cancer stem cells via the lncRNA-miRNA/protein axis.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {51},
pmid = {38600465},
issn = {1689-1392},
support = {DY135-B-04//China Ocean Mineral Resources Research and Development Association/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems ; Carcinogenesis/genetics ; Apoptosis/genetics ; Neoplastic Stem Cells/metabolism ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Methyltransferases/genetics ; },
abstract = {BACKGROUND: Long noncoding RNAs (lncRNAs) play essential roles in the tumorigenesis of gastric cancer. However, the influence of lncRNA methylation on gastric cancer stem cells (GCSCs) remains unclear.

METHODS: The N6-methyladenosine (m6A) levels of lncRNAs in gastric cancer stem cells were detected by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the results were validated by MeRIP-quantitative polymerase chain reaction (qPCR). Specific sites of m6A modification on lncRNAs were detected by single-base elongation- and ligation-based qPCR amplification (SELECT). By constructing and transfecting the plasmid expressing methyltransferase-like 3 (METTL3) fused with catalytically inactivated Cas13 (dCas13b) and guide RNA targeting specific methylation sites of lncRNAs, we obtained gastric cancer stem cells with site-specific methylation of lncRNAs. Reverse transcription (RT)-qPCR and Western blot were used for detecting the stemness of treated gastric cancer stem cells.

RESULTS: The site-specific methylation of PSMA3-AS1 and MIR22HG suppressed apoptosis and promoted stemness of GCSCs. LncRNA methylation enhanced the stability of PSMA3-AS1 and MIR22HG to suppress apoptosis of GCSCs via the PSMA3-AS1-miR-411-3p- or MIR22HG-miR-24-3p-SERTAD1 axis. Simultaneously, the methylated lncRNAs promoted the interaction between PSMA3-AS1 and the EEF1A1 protein or MIR22HG and the LRPPRC protein, stabilizing the proteins and leading to the suppression of apoptosis. The in vivo data revealed that the methylated PSMA3-AS1 and MIR22HG triggered tumorigenesis of GCSCs.

CONCLUSIONS: Our study revealed the requirement for site-specific methylation of lncRNAs in the tumorigenesis of GCSCs, contributing novel insights into cancer development.},
}

Humans
*MicroRNAs/genetics/metabolism
*RNA, Long Noncoding/genetics/metabolism
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems
Carcinogenesis/genetics
Apoptosis/genetics
Neoplastic Stem Cells/metabolism
Cell Proliferation/genetics
Gene Expression Regulation, Neoplastic
Methyltransferases/genetics

@article {pmid38600182,
year = {2024},
author = {Moncaut, N},
title = {Streamlining mouse genome editing by integrating AAV repair template delivery and CRISPR-Cas electroporation.},
journal = {Lab animal},
volume = {},
number = {},
pages = {},
pmid = {38600182},
issn = {1548-4475},
}

@article {pmid38599852,
year = {2024},
author = {Liu, S and Zhang, Y and Luo, Y and Liu, J},
title = {Traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine.},
journal = {Journal of diabetes},
volume = {16},
number = {4},
pages = {e13545},
pmid = {38599852},
issn = {1753-0407},
support = {20202ACBL206008//Natural Science Foundation of Jiangxi Provincial/ ; 81760150//National Natural Science Foundation of China/ ; 82160162//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Regenerative Medicine ; Pancreas ; *Diabetes Mellitus, Type 1 ; Hepatocytes ; },
abstract = {Although pancreas and islet cell transplantation are the only ways to prevent the late complications of insulin-dependent diabetes, a shortage of donors is a major obstacle to tissue and organ transplantation. Stem cell therapy is an effective treatment for diabetes and other pancreatic-related diseases, which can be achieved by inducing their differentiation into insulin-secreting cells. The liver is considered an ideal source of pancreatic cells due to its similar developmental origin and strong regenerative ability as the pancreas. This article reviews the traditional and emerging strategies using hepatocytes for pancreatic regenerative medicine and evaluates their advantages and challenges. Gene reprogramming and chemical reprogramming technologies are traditional strategies with potential to improve the efficiency and specificity of cell reprogramming and promote the transformation of hepatocytes into islet cells. At the same time, organoid technology, as an emerging strategy, has received extensive attention. Biomaterials provide a three-dimensional culture microenvironment for cells, which helps improve cell survival and differentiation efficiency. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has brought new opportunities and challenges to the development of organoid technology.},
}

Humans
*CRISPR-Cas Systems
Regenerative Medicine
Pancreas
*Diabetes Mellitus, Type 1
Hepatocytes

@article {pmid38599684,
year = {2024},
author = {Bukhari, H and Nithianadam, V and Battaglia, RA and Cicalo, A and Sarkar, S and Comjean, A and Hu, Y and Leventhal, MJ and Dong, X and Feany, MB},
title = {Transcriptional programs mediating neuronal toxicity and altered glial-neuronal signaling in a Drosophila knock-in tauopathy model.},
journal = {Genome research},
volume = {},
number = {},
pages = {},
doi = {10.1101/gr.278576.123},
pmid = {38599684},
issn = {1549-5469},
abstract = {Missense mutations in the gene encoding the microtubule-associated protein tau cause autosomal dominant forms of frontotemporal dementia. Multiple models of frontotemporal dementia based on transgenic expression of human tau in experimental model organisms, including Drosophila, have been described. These models replicate key features of the human disease, but do not faithfully recreate the genetic context of the human disorder. Here we use CRISPR-Cas mediated gene editing to model frontotemporal dementia caused by the tau P301L mutation by creating the orthologous mutation, P251L, in the endogenous Drosophila tau gene. Flies heterozygous or homozygous for tau P251L display age-dependent neurodegeneration, metabolic defects, and accumulate DNA damage in affected neurons. To understand the molecular events promoting neuronal dysfunction and death in knock-in flies we performed single-cell RNA sequencing on approximately 130,000 cells from brains of tau P251L mutant and control flies. We found that expression of disease-associated mutant tau altered gene expression cell autonomously in all neuronal cell types identified. Gene expression was also altered in glial cells, suggestive of non-cell autonomous regulation. Cell signaling pathways, including glial-neuronal signaling, were broadly dysregulated as were brain region and cell type-specific protein interaction networks and gene regulatory programs. In summary, we present here a genetic model of tauopathy, which faithfully recapitulates the genetic context and phenotypic features of the human disease and use the results of comprehensive single cell sequencing analysis to outline pathways of neurotoxicity and highlight the potential role of non-cell autonomous changes in glia.},
}

@article {pmid38598861,
year = {2024},
author = {Burbano, DA and Kiattisewee, C and Karanjia, AV and Cardiff, RAL and Faulkner, ID and Sugianto, W and Carothers, JM},
title = {CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications.},
journal = {Annual review of chemical and biomolecular engineering},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-chembioeng-100522-114706},
pmid = {38598861},
issn = {1947-5446},
abstract = {In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.},
}

@article {pmid38521283,
year = {2024},
author = {Hwang, J and Ye, DY and Jung, GY and Jang, S},
title = {Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.},
journal = {Biotechnology advances},
volume = {72},
number = {},
pages = {108343},
doi = {10.1016/j.biotechadv.2024.108343},
pmid = {38521283},
issn = {1873-1899},
mesh = {*Gene Editing ; *Genetic Engineering ; Mutagenesis ; Interspersed Repetitive Sequences ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.},
}

*Gene Editing
*Genetic Engineering
Mutagenesis
Interspersed Repetitive Sequences
CRISPR-Cas Systems/genetics

@article {pmid38490381,
year = {2024},
author = {Sun, Z and Wang, M and Wang, W and Li, D and Wang, J and Sui, G},
title = {Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly.},
journal = {International journal of biological macromolecules},
volume = {265},
number = {Pt 1},
pages = {130847},
doi = {10.1016/j.ijbiomac.2024.130847},
pmid = {38490381},
issn = {1879-0003},
mesh = {Animals ; Humans ; *Alphavirus/genetics/metabolism ; Capsid Proteins/genetics/metabolism ; RNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genomics ; Virion/genetics ; },
abstract = {Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.},
}

Animals
Humans
*Alphavirus/genetics/metabolism
Capsid Proteins/genetics/metabolism
RNA, Viral/genetics
RNA, Guide, CRISPR-Cas Systems
Genomics
Virion/genetics

@article {pmid38235591,
year = {2024},
author = {Ghahremani, S and Kanwal, A and Pettinato, A and Ladha, F and Legere, N and Thakar, K and Zhu, Y and Tjong, H and Wilderman, A and Stump, WT and Greenberg, L and Greenberg, MJ and Cotney, J and Wei, CL and Hinson, JT},
title = {CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.},
journal = {Circulation},
volume = {149},
number = {16},
pages = {1285-1297},
doi = {10.1161/CIRCULATIONAHA.123.063972},
pmid = {38235591},
issn = {1524-4539},
support = {R01 HL141086/HL/NHLBI NIH HHS/United States ; R01 HL165220/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Cardiomyopathy, Dilated/genetics/therapy/pathology ; Connectin/genetics ; Haploinsufficiency/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Myocytes, Cardiac/metabolism ; },
abstract = {BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity.

METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation.

RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts.

CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.},
}

Humans
*Cardiomyopathy, Dilated/genetics/therapy/pathology
Connectin/genetics
Haploinsufficiency/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems
Myocytes, Cardiac/metabolism

@article {pmid38598855,
year = {2024},
author = {Hsieh, SC and Peters, JE},
title = {Natural and Engineered Guide RNA-directed Transposition with CRISPR-Associated Tn7-like Transposons.},
journal = {Annual review of biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-biochem-030122-041908},
pmid = {38598855},
issn = {1545-4509},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.},
}

@article {pmid38597925,
year = {2024},
author = {Li, J and Yan, K and Wang, S and Wang, P and Jiao, J and Dong, Y},
title = {Alteration of the intestinal microbiota and serum metabolites in a mouse model of Pon1 gene ablation.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {38},
number = {7},
pages = {e23611},
doi = {10.1096/fj.202302344R},
pmid = {38597925},
issn = {1530-6860},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; RNA, Guide, CRISPR-Cas Systems ; Disease Models, Animal ; *Microbiota ; Aryldialkylphosphatase/genetics ; Mice, Knockout ; },
abstract = {Mutations in the Paraoxonase 1 (Pon1) gene underlie aging, cardiovascular disease, and impairments of the nervous and gastrointestinal systems and are linked to the intestinal microbiome. The potential role of Pon1 in modulating the intestinal microbiota and serum metabolites is poorly understood. The present study demonstrated that mice with genomic excision of Pon1 by a multiplexed guide RNA CRISPR/Cas9 approach exhibited disrupted gut microbiota, such as significantly depressed alpha-diversity and distinctly separated beta diversity, accompanied by varied profiles of circulating metabolites. Furthermore, genomic knock in of Pon1 exerted a distinct effect on the intestinal microbiome and serum metabolome, including dramatically enriched Aerococcus, linoleic acid and depleted Bacillus, indolelactic acid. Specifically, a strong correlation was established between bacterial alterations and metabolites in Pon1 knockout mice. In addition, we identified metabolites related to gut bacteria in response to Pon1 knock in. Thus, the deletion of Pon1 affects the gut microbiome and functionally modifies serum metabolism, which can lead to dysbiosis, metabolic dysfunction, and infection risk. Together, these findings put forth a role for Pon1 in microbial alterations that contribute to metabolism variations. The function of Pon1 in diseases might at least partially depend on the microbiome.},
}

Animals
Mice
*Gastrointestinal Microbiome/genetics
RNA, Guide, CRISPR-Cas Systems
Disease Models, Animal
*Microbiota
Aryldialkylphosphatase/genetics
Mice, Knockout

@article {pmid38597776,
year = {2024},
author = {Xie, H and Su, F and Niu, Q and Geng, L and Cao, X and Song, M and Dong, J and Zheng, Z and Guo, R and Zhang, Y and Deng, Y and Ji, Z and Pang, K and Zhu, JK and Zhu, J},
title = {Knockout of miR396 genes increases seed size and yield in soybean.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13660},
pmid = {38597776},
issn = {1744-7909},
support = {2021LZGC003//Seed-Industrialized Development Program in Shandong Province, China/ ; 2022AH010056//the Innovative research team of Anhui education/ ; 2021LZGC012//Key Research & Development Program of Shandong Province, China/ ; 32270367//National Natural Science Foundation of China/ ; HSPHDSRF-2023-11-007//the PhD Scientific Research and Innovation Foundation of Sanya Yazhou Bay Science and Technology City/ ; NK2022010301//National Key Research and Development Program of China/ ; },
abstract = {Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.},
}

@article {pmid38414349,
year = {2024},
author = {Zeng, J and Liang, X and Duan, L and Tan, F and Chen, L and Qu, J and Li, J and Li, K and Luo, D and Hu, Z},
title = {Targeted disruption of the BCR-ABL fusion gene by Cas9/dual-sgRNA inhibits proliferation and induces apoptosis in chronic myeloid leukemia cells.},
journal = {Acta biochimica et biophysica Sinica},
volume = {56},
number = {4},
pages = {525-537},
doi = {10.3724/abbs.2023280},
pmid = {38414349},
issn = {1745-7270},
mesh = {Humans ; Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems ; Fusion Proteins, bcr-abl/genetics/metabolism ; Genes, abl ; Proto-Oncogene Proteins c-bcr/genetics/metabolism ; CRISPR-Cas Systems ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/therapy ; Apoptosis/genetics ; Cell Proliferation/genetics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The BCR-ABL fusion gene, formed by the fusion of the breakpoint cluster region protein (BCR) and the Abl Oncogene 1, Receptor Tyrosine Kinase (ABL) genes, encodes the BCR-ABL oncoprotein, which plays a crucial role in leukemogenesis. Current therapies have limited efficacy in patients with chronic myeloid leukemia (CML) because of drug resistance or disease relapse. Identification of novel strategies to treat CML is essential. This study aims to explore the efficiency of novel CRISPR-associated protein 9 (Cas9)/dual-single guide RNA (sgRNA)-mediated disruption of the BCR-ABL fusion gene by targeting BCR and cABL introns. A co-expression vector for Cas9 green fluorescent protein (GFP)/dual-BA-sgRNA targeting BCR and cABL introns is constructed to produce lentivirus to affect BCR-ABL expression in CML cells. The effects of dual-sgRNA virus-mediated disruption of BCR-ABL are analyzed via the use of a genomic sequence and at the protein expression level. Cell proliferation, cell clonogenic ability, and cell apoptosis are assessed after dual sgRNA virus infection, and phosphorylated BCR-ABL and its downstream signaling molecules are detected. These effects are further confirmed in a CML mouse model via tail vein injection of Cas9-GFP/dual-BA-sgRNA virus-infected cells and in primary cells isolated from patients with CML. Cas9-GFP/dual-BA-sgRNA efficiently disrupts BCR-ABL at the genomic sequence and gene expression levels in leukemia cells, leading to blockade of the BCR-ABL tyrosine kinase signaling pathway and disruption of its downstream molecules, followed by cell proliferation inhibition and cell apoptosis induction. This method prolongs the lifespan of CML model mice. Furthermore, the effect is confirmed in primary cells derived from patients with CML.},
}

Humans
Animals
Mice
*RNA, Guide, CRISPR-Cas Systems
Fusion Proteins, bcr-abl/genetics/metabolism
Genes, abl
Proto-Oncogene Proteins c-bcr/genetics/metabolism
CRISPR-Cas Systems
*Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/metabolism/therapy
Apoptosis/genetics
Cell Proliferation/genetics
Drug Resistance, Neoplasm/genetics

@article {pmid38365083,
year = {2024},
author = {Duan, X and Li, H and Tan, X and Liu, N and Wang, X and Zhang, W and Liu, Y and Ma, W and Wu, Y and Ma, L and Fan, Y},
title = {Polygonum cillinerve polysaccharide inhibits transmissible gastroenteritis virus by regulating microRNA-181.},
journal = {Veterinary journal (London, England : 1997)},
volume = {304},
number = {},
pages = {106083},
doi = {10.1016/j.tvjl.2024.106083},
pmid = {38365083},
issn = {1532-2971},
mesh = {Animals ; Swine ; *Transmissible gastroenteritis virus/genetics ; *Polygonum/genetics ; RNA, Guide, CRISPR-Cas Systems ; Signal Transduction ; *MicroRNAs/genetics ; },
abstract = {Transmissible gastroenteritis virus (TGEV) is an important pathogen capable of altering the expression profile of cellular miRNA. In this study, the potential of Polygonum cillinerve polysaccharide (PCP) to treat TGEV-infected piglets was evaluated through in vivo experiments. High-throughput sequencing technology was employed to identify 9 up-regulated and 17 down-regulated miRNAs during PCP-mediated inhibition of TGEV infection in PK15 cells. Additionally, miR-181 was found to be associated with target genes of key proteins in the apoptosis pathway. PK15 cells were treated with various concentrations of PCP following transfection with miR-181 mimic or inhibitor. Real-time PCR assessed the impact on TGEV replication, while electron microscopy (TEM) and Hoechst fluorescence staining evaluated cellular functionality. Western blot analysis was utilized to assess the expression of key signaling factors-cytochrome C (cyt C), caspase 9, and P53-in the apoptotic signaling pathway. The results showed that compared with the control group, 250 μg/mL PCP significantly inhibited TGEV gRNA replication and gene N expression (P < 0.01). Microscopic examination revealed uniform cell morphology and fewer floating cells in PCP-treated groups (250 and 125 μg/mL). TEM analysis showed no typical virus structure in the 250 μg/mL PCP group, and apoptosis staining indicated a significant reduction in apoptotic cells at this concentration. Furthermore, PCP may inhibit TGEV-induced apoptosis via the Caspase-dependent mitochondrial pathway following miR-181 transfection. These findings provide a theoretical basis for further exploration into the mechanism of PCP's anti-TGEV properties.},
}

Animals
Swine
*Transmissible gastroenteritis virus/genetics
*Polygonum/genetics
RNA, Guide, CRISPR-Cas Systems
Signal Transduction
*MicroRNAs/genetics

@article {pmid38284432,
year = {2024},
author = {Bagheri, N and Chamorro, A and Idili, A and Porchetta, A},
title = {PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {63},
number = {17},
pages = {e202319677},
doi = {10.1002/anie.202319677},
pmid = {38284432},
issn = {1521-3773},
support = {MFAG 2022 - ID. 27151 project//Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; 101086341//H2020 Marie Skłodowska-Curie Actions/ ; MSCA_0000010//European Union NextGenerationEU/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Gene Editing/methods ; DNA/metabolism ; Nucleic Acid Conformation ; },
abstract = {The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems
Gene Editing/methods
DNA/metabolism
Nucleic Acid Conformation

@article {pmid38589794,
year = {2024},
author = {Wang, Y and Zhai, Y and Zhang, M and Song, C and Zhang, Y and Zhang, G},
title = {Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.},
journal = {Cellular & molecular biology letters},
volume = {29},
number = {1},
pages = {48},
pmid = {38589794},
issn = {1689-1392},
support = {31970666//the National Natural Science Foundation of China/ ; tsqn201812054//Taishan Scholar Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods

@article {pmid38587910,
year = {2024},
author = {Sakamoto, N and Watanabe, K and Awazu, A and Yamamoto, T},
title = {CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.},
journal = {Zoological science},
volume = {41},
number = {2},
pages = {159-166},
doi = {10.2108/zs230052},
pmid = {38587910},
issn = {0289-0003},
mesh = {Animals ; *Anthocidaris/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems ; Gene Knockout Techniques ; Sea Urchins/genetics ; Gene Editing/methods ; },
abstract = {Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.},
}

Animals
*Anthocidaris/genetics
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems
Gene Knockout Techniques
Sea Urchins/genetics
Gene Editing/methods

@article {pmid38585710,
year = {2024},
author = {Xiao, Y and Qin, T and He, S and Chen, Y and Li, H and He, Q and Wang, X and Yang, S},
title = {Systematic investigation of TetR-family transcriptional regulators and their roles on lignocellulosic inhibitor acetate tolerance in Zymomonas mobilis.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {12},
number = {},
pages = {1385519},
pmid = {38585710},
issn = {2296-4185},
abstract = {TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.},
}

@article {pmid38585687,
year = {2024},
author = {Fischer, J and Fedotova, A and Jaki, L and Sallard, E and Erhardt, A and Fuchs, J and Ruzsics, Z},
title = {Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries.},
journal = {Molecular therapy. Methods & clinical development},
volume = {32},
number = {2},
pages = {101241},
pmid = {38585687},
issn = {2329-0501},
abstract = {While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 10[6] unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 10[4] unique rAds per cm[2] of transfected cell culture.},
}

@article {pmid38584940,
year = {2024},
author = {Carhuaricra-Huaman, D and Gonzalez, IHL and Ramos, PL and da Silva, AM and Setubal, JC},
title = {Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths (Bradypus variegatus), the first from a non-human host.},
journal = {PeerJ},
volume = {12},
number = {},
pages = {e17206},
pmid = {38584940},
issn = {2167-8359},
mesh = {Animals ; *Sloths/genetics ; Phylogeny ; Brazil ; *Alcaligenaceae/genetics ; },
abstract = {Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.},
}

Animals
*Sloths/genetics
Phylogeny
Brazil
*Alcaligenaceae/genetics

@article {pmid38583547,
year = {2024},
author = {Saunier, M and Fortier, LC and Soutourina, O},
title = {RNA-based regulation in bacteria-phage interactions.},
journal = {Anaerobe},
volume = {87},
number = {},
pages = {102851},
doi = {10.1016/j.anaerobe.2024.102851},
pmid = {38583547},
issn = {1095-8274},
abstract = {Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.},
}

@article {pmid38582932,
year = {2024},
author = {Wang, Y and Peng, Y and Zi, G and Chen, J and Peng, B},
title = {Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {8095},
pmid = {38582932},
issn = {2045-2322},
mesh = {Humans ; Female ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; RNA, Messenger/genetics/metabolism ; *Breast Neoplasms/genetics ; Gene Editing/methods ; },
abstract = {Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.},
}

Humans
Female
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger/genetics/metabolism
*Breast Neoplasms/genetics
Gene Editing/methods

@article {pmid38582885,
year = {2024},
author = {Chan, YT and Wu, J and Lu, Y and Li, Q and Feng, Z and Xu, L and Yuan, H and Xing, T and Zhang, C and Tan, HY and Feng, Y and Wang, N},
title = {Loss of lncRNA LINC01056 leads to sorafenib resistance in HCC.},
journal = {Molecular cancer},
volume = {23},
number = {1},
pages = {74},
pmid = {38582885},
issn = {1476-4598},
support = {104006600//University Research Committee of The University of Hong Kong/ ; 17119621//Research Grant Committee of Hong Kong/ ; 19201591//The Health and Medical Research Fund/ ; PRP/028/22FX//Innovation and Technology Fund/ ; },
mesh = {Humans ; Mice ; Animals ; Sorafenib/pharmacology/therapeutic use ; *Carcinoma, Hepatocellular/drug therapy/genetics/metabolism ; *RNA, Long Noncoding/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; PPAR alpha/genetics/metabolism/therapeutic use ; Proteomics ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND AND AIMS: Sorafenib is a major nonsurgical option for patients with advanced hepatocellular carcinoma (HCC); however, its clinical efficacy is largely undermined by the acquisition of resistance. The aim of this study was to identify the key lncRNA involved in the regulation of the sorafenib response in HCC.

MATERIALS AND METHODS: A clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) single-guide RNA (sgRNA) synergistic activation mediator (SAM)-pooled lncRNA library was applied to screen for the key lncRNA regulated by sorafenib treatment. The role of the identified lncRNA in mediating the sorafenib response in HCC was examined in vitro and in vivo. The underlying mechanism was delineated by proteomic analysis. The clinical significance of the expression of the identified lncRNA was evaluated by multiplex immunostaining on a human HCC microtissue array.

RESULTS: CRISPR/Cas9 lncRNA library screening revealed that Linc01056 was among the most downregulated lncRNAs in sorafenib-resistant HCC cells. Knockdown of Linc01056 reduced the sensitivity of HCC cells to sorafenib, suppressing apoptosis in vitro and promoting tumour growth in mice in vivo. Proteomic analysis revealed that Linc01056 knockdown in sorafenib-treated HCC cells induced genes related to fatty acid oxidation (FAO) while repressing glycolysis-associated genes, leading to a metabolic switch favouring higher intracellular energy production. FAO inhibition in HCC cells with Linc01056 knockdown significantly restored sensitivity to sorafenib. Mechanistically, we determined that PPARα is the critical molecule governing the metabolic switch upon Linc01056 knockdown in HCC cells and indeed, PPARα inhibition restored the sorafenib response in HCC cells in vitro and HCC tumours in vivo. Clinically, Linc01056 expression predicted optimal overall and progression-free survival outcomes in HCC patients and predicted a better sorafenib response. Linc01056 expression indicated a low FAO level in HCC.

CONCLUSION: Our study identified Linc01056 as a critical epigenetic regulator and potential therapeutic target in the regulation of the sorafenib response in HCC.},
}

Humans
Mice
Animals
Sorafenib/pharmacology/therapeutic use
*Carcinoma, Hepatocellular/drug therapy/genetics/metabolism
*RNA, Long Noncoding/genetics
*Liver Neoplasms/drug therapy/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems
PPAR alpha/genetics/metabolism/therapeutic use
Proteomics
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Gene Expression Regulation, Neoplastic

@article {pmid38565025,
year = {2024},
author = {Zhu, Y and Lin, Y and Gong, B and Zhang, Y and Su, G and Yu, Y},
title = {Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping.},
journal = {Biosensors & bioelectronics},
volume = {255},
number = {},
pages = {116255},
doi = {10.1016/j.bios.2024.116255},
pmid = {38565025},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genotype ; Polymorphism, Single Nucleotide/genetics ; *Biosensing Techniques ; Apolipoproteins E ; Uracil-DNA Glycosidase ; },
abstract = {Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.},
}

Humans
*CRISPR-Cas Systems/genetics
Genotype
Polymorphism, Single Nucleotide/genetics
*Biosensing Techniques
Apolipoproteins E
Uracil-DNA Glycosidase

@article {pmid38552526,
year = {2024},
author = {Yang, Y and Sun, L and Zhao, J and Jiao, Y and Han, T and Zhou, X},
title = {Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5'-overhang.},
journal = {Biosensors & bioelectronics},
volume = {255},
number = {},
pages = {116239},
doi = {10.1016/j.bios.2024.116239},
pmid = {38552526},
issn = {1873-4235},
mesh = {*RNA, Guide, CRISPR-Cas Systems ; *Biosensing Techniques ; RNA ; Recombinases ; SARS-CoV-2 ; CRISPR-Cas Systems/genetics ; },
abstract = {The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5'7U LbuCas13a crRNA, where the 5'-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5'-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription-recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.},
}

*RNA, Guide, CRISPR-Cas Systems
*Biosensing Techniques
RNA
Recombinases
SARS-CoV-2
CRISPR-Cas Systems/genetics

@article {pmid38504114,
year = {2024},
author = {Yao, D and Tycko, J and Oh, JW and Bounds, LR and Gosai, SJ and Lataniotis, L and Mackay-Smith, A and Doughty, BR and Gabdank, I and Schmidt, H and Guerrero-Altamirano, T and Siklenka, K and Guo, K and White, AD and Youngworth, I and Andreeva, K and Ren, X and Barrera, A and Luo, Y and Yardımcı, GG and Tewhey, R and Kundaje, A and Greenleaf, WJ and Sabeti, PC and Leslie, C and Pritykin, Y and Moore, JE and Beer, MA and Gersbach, CA and Reddy, TE and Shen, Y and Engreitz, JM and Bassik, MC and Reilly, SK},
title = {Multicenter integrated analysis of noncoding CRISPRi screens.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {723-734},
pmid = {38504114},
issn = {1548-7105},
support = {UM1 HG012053/HG/NHGRI NIH HHS/United States ; R00 HG009917/HG/NHGRI NIH HHS/United States ; U01 HG012103/HG/NHGRI NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; R01 HG010741/HG/NHGRI NIH HHS/United States ; R00 HG010669/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; UM1 HG009428/HG/NHGRI NIH HHS/United States ; U24 HG009397/HG/NHGRI NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; U01 HG009380/HG/NHGRI NIH HHS/United States ; U01 HG009395/HG/NHGRI NIH HHS/United States ; U01 HG009431/HG/NHGRI NIH HHS/United States ; K00 DK126120/DK/NIDDK NIH HHS/United States ; UM1 HG009435/HG/NHGRI NIH HHS/United States ; R01 HG012872/HG/NHGRI NIH HHS/United States ; UM1 HG009436/HG/NHGRI NIH HHS/United States ; U24 HG009446/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Guide, CRISPR-Cas Systems ; Genome ; K562 Cells ; },
abstract = {The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.},
}

Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Guide, CRISPR-Cas Systems
Genome
K562 Cells

@article {pmid38499809,
year = {2024},
author = {Manjunath, LE and Singh, A and Devi Kumar, S and Vasu, K and Kar, D and Sellamuthu, K and Eswarappa, SM},
title = {Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {2118-2143},
pmid = {38499809},
issn = {1469-3178},
support = {BT/PR38405/GET/119/309/2020//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; SB/SJF/2020-21/18//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; STARS/APR2019/BS/328/FS//Ministry of Education, Government of India/ ; ID-2021-13048//Indian Council of Medical Research (ICMR)/ ; },
mesh = {Animals ; Codon, Terminator/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *RNA, Guide, CRISPR-Cas Systems ; Codon, Nonsense/genetics ; RNA, Messenger/genetics/metabolism ; Protein Biosynthesis ; Mammals/genetics/metabolism ; },
abstract = {Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.},
}

Animals
Codon, Terminator/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*RNA, Guide, CRISPR-Cas Systems
Codon, Nonsense/genetics
RNA, Messenger/genetics/metabolism
Protein Biosynthesis
Mammals/genetics/metabolism

@article {pmid38490564,
year = {2024},
author = {Pennati, A and Jakobi, M and Zeng, F and Ciampa, L and Rothbächer, U},
title = {Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.},
journal = {Developmental biology},
volume = {510},
number = {},
pages = {31-39},
doi = {10.1016/j.ydbio.2024.03.003},
pmid = {38490564},
issn = {1095-564X},
mesh = {Animals ; *Ciona intestinalis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems ; *Ciona/genetics ; Electroporation ; Gene Editing/methods ; },
abstract = {CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.},
}

Animals
*Ciona intestinalis/genetics/metabolism
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems
*Ciona/genetics
Electroporation
Gene Editing/methods

@article {pmid38412306,
year = {2024},
author = {Capin, J and Harrison, A and Raele, RA and Yadav, SKN and Baiwir, D and Mazzucchelli, G and Quinton, L and Satchwell, TJ and Toye, AM and Schaffitzel, C and Berger, I and Aulicino, F},
title = {An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.},
journal = {Nucleic acids research},
volume = {52},
number = {6},
pages = {3450-3468},
pmid = {38412306},
issn = {1362-4962},
support = {834631/ERC_/European Research Council/International ; MR/V010506/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; Mammals/genetics ; Baculoviridae/genetics ; DNA/genetics ; },
abstract = {CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.},
}

Animals
Humans
*Gene Editing
*CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
Mammals/genetics
Baculoviridae/genetics
DNA/genetics

@article {pmid38409225,
year = {2024},
author = {Montero, JJ and Trozzo, R and Sugden, M and Öllinger, R and Belka, A and Zhigalova, E and Waetzig, P and Engleitner, T and Schmidt-Supprian, M and Saur, D and Rad, R},
title = {Genome-scale pan-cancer interrogation of lncRNA dependencies using CasRx.},
journal = {Nature methods},
volume = {21},
number = {4},
pages = {584-596},
pmid = {38409225},
issn = {1548-7105},
support = {DFG RA1629/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB1371//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; (70114314)//Deutsche Krebshilfe (German Cancer Aid)/ ; Consolidator grant PACA-MET (819642)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; MSCA-ITNETN (861196//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; Long-Term Fellowship (ALFT 655-2019).//European Molecular Biology Organization (EMBO)/ ; },
mesh = {Humans ; Gene Expression Profiling ; *RNA, Long Noncoding/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Transcriptome ; *Neoplasms/genetics/metabolism ; },
abstract = {Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.},
}

Humans
Gene Expression Profiling
*RNA, Long Noncoding/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems
Transcriptome
*Neoplasms/genetics/metabolism

@article {pmid38379122,
year = {2024},
author = {Li, S and Mei, L and He, C and Cai, X and Wu, H and Wu, X and Liu, Y and Feng, Y and Song, J},
title = {Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.},
journal = {Human cell},
volume = {37},
number = {3},
pages = {817-831},
pmid = {38379122},
issn = {1749-0774},
support = {82071065//National Natural Science Foundation of China/ ; 2014CB541702//Major State Basic Research Development Program of China/ ; 2020YFC2005204//Key Technologies Research and Development Program/ ; 2020SK2106//Key Research and Development Program of Hunan Province of China/ ; 2021zzts0345//Fundamental Research Funds for Central Universities of the Central South University/ ; CX20210355//Fundamental Research Funds for Central Universities of the Central South University/ ; 2021M693566//Postdoctoral Research Foundation of China/ ; 2021T140751//Postdoctoral Research Foundation of China/ ; 2020RC2013//Science and Technology Program of Hunan Province/ ; 2021JJ41017//Natural Science Foundation of Hunan Province/ ; 2021JJ31084//Natural Science Foundation of Hunan Province/ ; 202107010047//Health Commission of Hunan Province/ ; },
mesh = {Humans ; *Osteogenesis Imperfecta/genetics/therapy ; Collagen Type I/genetics ; *Induced Pluripotent Stem Cells ; Leukocytes, Mononuclear ; CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; Mutation ; },
abstract = {Van der Hoeve's syndrome, also known as osteogenesis imperfecta (OI), is a genetic connective tissue disorder characterized by fragile, fracture-prone bone and hearing loss. The disease is caused by a gene mutation in one of the two type I collagen genes COL1A1 or COL1A2. In this study, we identified a novel frameshift mutation of the COL1A1 gene (c.1607delG) in a family with OI using whole-exome sequencing, bioinformatics analysis and Sanger sequencing. This mutation may lead to the deletion of a portion of exon 23 and the generation of a premature stop codon in the COL1A1 gene. To further investigate the impact of this mutation, we established two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells of OI patients carrying a novel mutation in the COL1A1 gene. Osteoblasts (OB) derived from OI-iPSCs exhibited reduced production of type I collagen and diminished ability to differentiate into osteoblasts. Using a CRISPR-based homology-directed repair strategy, we corrected the OI disease-causing COL1A1 novel mutations in iPSCs generated from an affected individual. Our results demonstrated that the diminished expression of type I collagen and osteogenic potential were enhanced in OB induced from corrected OI-iPSCs compared to those from OI-iPSCs. Overall, our results provide new insights into the genetic basis of Van der Hoeve's syndrome and highlight the potential of iPSC technology for disease modeling and therapeutic development.},
}

Humans
*Osteogenesis Imperfecta/genetics/therapy
Collagen Type I/genetics
*Induced Pluripotent Stem Cells
Leukocytes, Mononuclear
CRISPR-Cas Systems/genetics
Collagen Type I, alpha 1 Chain
Mutation

@article {pmid38347223,
year = {2024},
author = {Krink, N and Nikel, PI and Beisel, CL},
title = {A Hitchhiker's guide to CRISPR editing tools in bacteria : CRISPR can help unlock the bacterial world, but technical and regulatory barriers persist.},
journal = {EMBO reports},
volume = {25},
number = {4},
pages = {1694-1699},
pmid = {38347223},
issn = {1469-3178},
support = {NNF20OC0065068//Novo Nordisk Fonden (NNF)/ ; NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF18OC0034818//Novo Nordisk Fonden (NNF)/ ; NNF21OC0067996//Novo Nordisk Fonden (NNF)/ ; 814418//EC | Horizon 2020 Framework Programme (H2020)/ ; 865973//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Bacteria/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. [Image: see text]},
}

*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Bacteria/genetics
Gene Editing
CRISPR-Cas Systems

@article {pmid38293815,
year = {2024},
author = {Wang, H and Tan, HY and Lian, J and Zhou, K},
title = {Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.},
journal = {Biotechnology and bioengineering},
volume = {121},
number = {5},
pages = {1543-1553},
doi = {10.1002/bit.28664},
pmid = {38293815},
issn = {1097-0290},
support = {LR20B060003//Natural Science Foundation of Zhejiang Province/ ; 22278361//Natural Science Foundation of China/ ; 226-2023-00015//Fundamental Research Funds for the Central Universities/ ; 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; 226-2023-00085//Fundamental Research Funds for the Central Universities/ ; //National Research Foundation of Singapore/ ; //Ministry of Education of Singapore/ ; //China Scholarship Council (CSC)/ ; //National University of Singapore/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics/metabolism ; Transcriptional Activation ; *Nanopore Sequencing ; Cloning, Molecular ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow. In this study, an in-house nanopore sequencing workflow was established for assessing the current methods of gRNA pool construction. The bias of pool construction was reduced by employing the polymerase-mediated non-amplifying method. Then, a small gRNA pool was utilized to characterize stronger activation domains, specifically MED2 (a subunit of mediator complex) and HAP4 (a heme activator protein), as well as to identify better gRNA choices for dCas12a-based gene activation in Saccharomyces cerevisiae. Furthermore, based on the better CRISPRa tool identified in this study, a custom gRNA pool, which consisted of 99 gRNAs targeting central metabolic pathways, was designed and employed to screen for gene targets that could improve ethanol utilization in S. cerevisiae. The nanopore sequencing-based workflow demonstrated here should provide a cost-effective approach for assessing the quality of customized gRNA library, leading to faster and more efficient genetic and metabolic engineering in S. cerevisiae.},
}

*RNA, Guide, CRISPR-Cas Systems
Saccharomyces cerevisiae/genetics/metabolism
Transcriptional Activation
*Nanopore Sequencing
Cloning, Molecular
CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid38199243,
year = {2024},
author = {Song, N and Fan, X and Guo, X and Tang, J and Li, H and Tao, R and Li, F and Li, J and Yang, D and Yao, C and Liu, P},
title = {A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for Synergistic Cancer Therapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {36},
number = {15},
pages = {e2309534},
doi = {10.1002/adma.202309534},
pmid = {38199243},
issn = {1521-4095},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; //Innovative Research Team of High-level Local Universities in Shanghai/ ; },
mesh = {Mice ; Animals ; *Photochemotherapy ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; Antioxidants ; Hemin ; Hydrogen Peroxide ; RNA, Guide, CRISPR-Cas Systems ; *Nanoparticles/therapeutic use ; Photosensitizing Agents/pharmacology/therapeutic use ; Hypoxia ; Cell Line, Tumor ; *Neoplasms/drug therapy ; },
abstract = {Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H2O2 to O2, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O2 to [1]O2. The integration of gene editing and PDT allows substantial accumulation of [1]O2 in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.},
}

Mice
Animals
*Photochemotherapy
CRISPR-Cas Systems
Reactive Oxygen Species/metabolism
Antioxidants
Hemin
Hydrogen Peroxide
RNA, Guide, CRISPR-Cas Systems
*Nanoparticles/therapeutic use
Photosensitizing Agents/pharmacology/therapeutic use
Hypoxia
Cell Line, Tumor
*Neoplasms/drug therapy

@article {pmid38164916,
year = {2024},
author = {Yaylacıoğlu Tuncay, F and Talim, B and Dinçer, PR},
title = {Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea.},
journal = {Current eye research},
volume = {49},
number = {5},
pages = {458-466},
doi = {10.1080/02713683.2023.2298904},
pmid = {38164916},
issn = {1460-2202},
mesh = {Mice ; Animals ; Humans ; Transforming Growth Factor beta/genetics/metabolism ; Zebrafish/genetics ; RNA, Guide, CRISPR-Cas Systems ; Cornea/metabolism ; *Corneal Dystrophies, Hereditary/genetics ; Mutation ; *Corneal Opacity/metabolism ; Extracellular Matrix Proteins/genetics/metabolism ; DNA Mutational Analysis ; Pedigree ; },
abstract = {PURPOSE: Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs.

METHODS: The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.

RESULTS: We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age.

CONCLUSION: This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.},
}

Mice
Animals
Humans
Transforming Growth Factor beta/genetics/metabolism
Zebrafish/genetics
RNA, Guide, CRISPR-Cas Systems
Cornea/metabolism
*Corneal Dystrophies, Hereditary/genetics
Mutation
*Corneal Opacity/metabolism
Extracellular Matrix Proteins/genetics/metabolism
DNA Mutational Analysis
Pedigree

@article {pmid38582601,
year = {2024},
author = {Koo, J and Zhu, GH and Palli, SR},
title = {CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105839},
doi = {10.1016/j.pestbp.2024.105839},
pmid = {38582601},
issn = {1095-9939},
mesh = {Animals ; RNA Interference ; Spodoptera/genetics/metabolism ; *CRISPR-Cas Systems ; *RNA, Double-Stranded/genetics/metabolism ; Insecta/genetics ; Larva/genetics/metabolism ; },
abstract = {Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.},
}

Animals
RNA Interference
Spodoptera/genetics/metabolism
*CRISPR-Cas Systems
*RNA, Double-Stranded/genetics/metabolism
Insecta/genetics
Larva/genetics/metabolism

@article {pmid38582582,
year = {2024},
author = {Li, JL and Li, SS and Luo, ZJ and Lu, J and Cai, XM and Luo, ZX and Bian, L and Xiu, CL and Fu, NX and Liu, NY and Li, ZQ},
title = {CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.},
journal = {Pesticide biochemistry and physiology},
volume = {200},
number = {},
pages = {105810},
doi = {10.1016/j.pestbp.2024.105810},
pmid = {38582582},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics ; Melanins/genetics ; CRISPR-Cas Systems ; Larva/genetics ; Pigmentation/genetics ; },
abstract = {Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.},
}

Animals
*Moths/genetics
Melanins/genetics
CRISPR-Cas Systems
Larva/genetics
Pigmentation/genetics

@article {pmid38581602,
year = {2024},
author = {Zhang, H and Liang, M and Chen, J and Wang, H and Ma, L},
title = {Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.},
journal = {Planta},
volume = {259},
number = {5},
pages = {112},
pmid = {38581602},
issn = {1432-2048},
support = {LY24C130004//Natural Science Foundation of Zhejiang Province/ ; LY22C135104//Natural Science Foundation of Zhejiang Province/ ; LY23C130002//Natural Science Foundation of Zhejiang Province/ ; 31501288//National Natural Science Foundation of China/ ; 20210207//Open Project Program of State Key Laboratory of Rice Biology/ ; CPSIBRF-CNRRI-202306//Central Public-interest Scientific Institution Basal Research Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *Oryza/genetics ; Disease Resistance/genetics ; Odorants ; Temperature ; Plant Breeding ; },
abstract = {The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.},
}

*CRISPR-Cas Systems
*Oryza/genetics
Disease Resistance/genetics
Odorants
Temperature
Plant Breeding

@article {pmid38580329,
year = {2024},
author = {Lo Presti, V and Meringa, A and Dunnebach, E and van Velzen, A and Moreira, AV and Stam, RW and Kotecha, RS and Krippner-Heidenreich, A and Heidenreich, OT and Plantinga, M and Cornel, A and Sebestyen, Z and Kuball, J and van Til, NP and Nierkens, S},
title = {Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.},
journal = {Journal for immunotherapy of cancer},
volume = {12},
number = {4},
pages = {},
pmid = {38580329},
issn = {2051-1426},
mesh = {Humans ; Child ; CD8-Positive T-Lymphocytes ; CRISPR-Cas Systems/genetics ; Fetal Blood ; Receptors, Antigen, T-Cell/genetics ; *Leukemia, Myeloid, Acute/genetics/therapy ; Cell Line, Tumor ; *Antineoplastic Agents ; Recurrence ; },
abstract = {BACKGROUND: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft.

METHODS: We produced CB-CD8[+] T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα (TRAC) and TCRβ (TRBC) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts.

RESULTS: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR[-/-] WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR[+/+] WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR[-/-] WT1-TCR CD8[+] CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells.

CONCLUSIONS: In summary, we show the feasibility of developing a potent CB-derived CD8[+] T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.},
}

Humans
Child
CD8-Positive T-Lymphocytes
CRISPR-Cas Systems/genetics
Fetal Blood
Receptors, Antigen, T-Cell/genetics
*Leukemia, Myeloid, Acute/genetics/therapy
Cell Line, Tumor
*Antineoplastic Agents
Recurrence

@article {pmid38579680,
year = {2024},
author = {Cavazzana, M and Miccio, A},
title = {The difficult translational pathway from animal models to patients.},
journal = {Cell stem cell},
volume = {31},
number = {4},
pages = {435-436},
doi = {10.1016/j.stem.2024.03.010},
pmid = {38579680},
issn = {1875-9777},
mesh = {Animals ; Humans ; *Gene Editing ; Hematopoietic Stem Cells/metabolism ; Models, Animal ; CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cell Transplantation ; },
abstract = {Lee et al.[1] analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.},
}

Animals
Humans
*Gene Editing
Hematopoietic Stem Cells/metabolism
Models, Animal
CRISPR-Cas Systems/genetics
*Hematopoietic Stem Cell Transplantation

@article {pmid38579669,
year = {2024},
author = {Jalil, S and Keskinen, T and Juutila, J and Sartori Maldonado, R and Euro, L and Suomalainen, A and Lapatto, R and Kuuluvainen, E and Hietakangas, V and Otonkoski, T and Hyvönen, ME and Wartiovaara, K},
title = {Genetic and functional correction of argininosuccinate lyase deficiency using CRISPR adenine base editors.},
journal = {American journal of human genetics},
volume = {111},
number = {4},
pages = {714-728},
doi = {10.1016/j.ajhg.2024.03.004},
pmid = {38579669},
issn = {1537-6605},
mesh = {Humans ; *Argininosuccinic Aciduria/genetics ; Argininosuccinate Lyase/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems ; Urea ; },
abstract = {Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.},
}

Humans
*Argininosuccinic Aciduria/genetics
Argininosuccinate Lyase/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Guide, CRISPR-Cas Systems
Urea

@article {pmid38576085,
year = {2024},
author = {Song, J and Luo, N and Dong, L and Peng, J and Yi, C},
title = {RNA base editors: The emerging approach of RNA therapeutics.},
journal = {Wiley interdisciplinary reviews. RNA},
volume = {15},
number = {2},
pages = {e1844},
doi = {10.1002/wrna.1844},
pmid = {38576085},
issn = {1757-7012},
support = {2023YFC3402200//Ministry of Science and Technology of the People's Republic of China/ ; 2019YFA0802200//Ministry of Science and Technology of the People's Republic of China/ ; 92153303//National Natural Science Foundation of China/ ; 21825701//National Natural Science Foundation of China/ ; Z231100002723005//Beijing Municipal Science and Technology Commission/ ; },
mesh = {*RNA/genetics ; *CRISPR-Cas Systems ; Gene Editing ; Oligonucleotides, Antisense ; RNA Interference ; },
abstract = {RNA-based therapeutics offer a flexible and reversible approach for treating genetic disorders, such as antisense oligonucleotides, RNA interference, aptamers, mRNA vaccines, and RNA editing. In recent years, significant advancements have been made in RNA base editing to correct disease-relevant point mutations. These achievements have significantly influenced the fields of biotechnology, biomedical research and therapeutics development. In this article, we provide a comprehensive overview of the design and performance of contemporary RNA base editors, including A-to-I, C-to-U, A-to-m[6]A, and U-to-Ψ. We compare recent innovative developments and highlight their applications in disease-relevant contexts. Lastly, we discuss the limitations and future prospects of utilizing RNA base editing for therapeutic purposes. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Development.},
}

*RNA/genetics
*CRISPR-Cas Systems
Gene Editing
Oligonucleotides, Antisense
RNA Interference