@article {pmid39628591,
year = {2024},
author = {Le, Y and Zhang, M and Wu, P and Wang, H and Ni, J},
title = {Biofuel production from lignocellulose via thermophile-based consolidated bioprocessing.},
journal = {Engineering microbiology},
volume = {4},
number = {4},
pages = {100174},
pmid = {39628591},
issn = {2667-3703},
abstract = {The depletion of fossil fuels and their impact on the environment have led to efforts to develop alternative sustainable fuels. While biofuel derived from lignocellulose is considered a sustainable, renewable, and green energy source, enhancing biofuel production and achieving a cost-effective bioconversion of lignocellulose at existing bio-refineries remains a challenge. Consolidated bioprocessing (CBP) using thermophiles can simplify this operation by integrating multiple processes, such as hydrolytic enzyme production, lignocellulose degradation, biofuel fermentation, and product distillation. This paper reviews recent developments in the conversion of lignocellulose to biofuel using thermophile-based CBP. First, advances in thermostable enzyme and thermophilic lignocellulolytic microorganism discovery and development for lignocellulosic biorefinery use are outlined. Then, several thermophilic CBP candidates and thermophilic microbes engineered to drive CBP of lignocellulose are reviewed. CRISPR/Cas-based genome editing tools developed for thermophiles are also highlighted. The potential applications of the Design-Build-Test-Learn (DBTL) synthetic biology strategy for designing and constructing thermophilic CBP hosts are also discussed in detail. Overall, this review illustrates how to develop highly sophisticated thermophilic CBP hosts for use in lignocellulosic biorefinery applications.},
}

@article {pmid39628447,
year = {2024},
author = {Carlsen, FM and Westberg, I and Johansen, IE and Andreasson, E and Petersen, BL},
title = {Strategies and Protocols for Optimized Genome Editing in Potato.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0068},
pmid = {39628447},
issn = {2573-1602},
abstract = {The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.},
}

@article {pmid39628369,
year = {2024},
author = {Kanitchinda, S and Sritunyalucksana, K and Chaijarasphong, T},
title = {Multiplex CRISPR-Cas Assay for Rapid, Isothermal and Visual Detection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimp.},
journal = {Journal of fish diseases},
volume = {},
number = {},
pages = {e14059},
doi = {10.1111/jfd.14059},
pmid = {39628369},
issn = {1365-2761},
support = {//Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)) (Grant no. FF-056/2566)/ ; },
abstract = {White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR-Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR-Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.},
}

@article {pmid39627841,
year = {2024},
author = {Sledzinski, P and Nowaczyk, M and Smielowska, MI and Olejniczak, M},
title = {CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair.},
journal = {BMC biology},
volume = {22},
number = {1},
pages = {282},
pmid = {39627841},
issn = {1741-7007},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; *Huntingtin Protein/genetics/metabolism ; Recombinational DNA Repair ; Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion/genetics ; DNA Repair ; },
abstract = {BACKGROUND: The expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences.

RESULTS: The location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in ~ 90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and DNA end resection. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using proteomic analysis, we identified novel factors that may be involved in CAG sequence repair.

CONCLUSIONS: Our study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.},
}

Humans
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems
*Huntingtin Protein/genetics/metabolism
Recombinational DNA Repair
Trinucleotide Repeats/genetics
Trinucleotide Repeat Expansion/genetics
DNA Repair

@article {pmid39626678,
year = {2024},
author = {Aviram, N and Shilton, AK and Lyn, NG and Reis, BS and Brivanlou, A and Marraffini, LA},
title = {Cas10 relieves host growth arrest to facilitate spacer retention during type III-A CRISPR-Cas immunity.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2024.11.005},
pmid = {39626678},
issn = {1934-6069},
abstract = {Cells from all kingdoms of life can enter growth arrest in unfavorable environmental conditions. Key to this process are mechanisms enabling recovery from this state. Staphylococcal type III-A CRISPR-Cas loci encode the Cas10 complex that uses a guide RNA to locate complementary viral transcripts and start an immune response. When the target sequence is expressed late in the viral lytic cycle, defense requires the activity of Csm6, a non-specific RNase that inhibits the growth of the infected cell. How Csm6 protects from infection and whether growth can be restored is not known. Here, we show that growth arrest provides immunity at the population level, preventing viral replication and allowing uninfected cells to propagate. In addition, the ssDNase activity of Cas10 is required for the regrowth of a subset of the arrested cells and the recovery of the infected host, presumably ending the immune response through degradation of the viral DNA.},
}

@article {pmid39624314,
year = {2024},
author = {Panahi, B and Rostampour, M and Ghaffari, MR and Nami, Y},
title = {Genome mining approach reveals the CRISPR-Cas systems features and characteristics in Lactobacillus delbrueckii strains.},
journal = {Heliyon},
volume = {10},
number = {22},
pages = {e39920},
pmid = {39624314},
issn = {2405-8440},
abstract = {This study employed a comprehensive genome-mining approach to characterize CRISPR-Cas systems in Lactobacillus delbrueckii strains. The analysis involved retrieving 105 genome sequences to explore the variety, occurrence, and evolution of CRISPR-Cas systems within the species. Homology analysis of spacer sequences in detected CRISPR arrays was conducted to assess the variety of target phages and plasmids. The evolutionary trajectories of spaceromes in each subtype of CRISPR arrays were determined by analyzing acquisition and deletion events under the selection pressure of foreign plasmids and phages. Among the analyzed strains, 53 contained only one CRISPR-Cas locus, 11 had two loci, and 21 featured three loci with complete CRISPR-Cas systems. Subtype designation of the results of current study revealed that 56 % of these systems belong to subtypes I-E/I-C, 23 % to subtypes III-A/III-D, and 17 % to subtype II-A. Notably, certain plasmids were found to be specifically targeted by distinct CRISPR array systems. The comparison of spacer sequences with phage genomes indicated that strains containing only CRISPR-Cas type-I systems targeted a broader range of phages. In conclusion, this study highlights the diversity of the three identified CRISPR-Cas types in L. delbrueckii strains and emphasizes their significant role in defense against phage invasion.},
}

@article {pmid39623998,
year = {2024},
author = {Wu, Y and Zhang, X and Li, J and Xie, J and Wang, L and Sun, H},
title = {[Progress of researches on toxoplasmosis vaccines based on the CRISPR/Cas9 technology].},
journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control},
volume = {36},
number = {5},
pages = {542-547},
doi = {10.16250/j.32.1374.2024026},
pmid = {39623998},
issn = {1005-6661},
support = {ZR2022MH271//Natural Science Foundation of Shandong Province/ ; 202301011242//Shandong Provincial Medical and Health Science and Technology Project/ ; 202101050153//Shandong Provincial Science and Technology Development Project for Medical Sciences and Health/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Toxoplasma/genetics/immunology ; *Toxoplasmosis/prevention & control/immunology/parasitology ; Protozoan Vaccines/immunology ; Gene Editing/methods ; },
abstract = {Toxoplasma gondii is an obligatory intracellular parasite which infects a variety of warm-blooded animals and causes toxoplasmosis. Toxoplasmosis seriously endangers human health and animal husbandry production. As one of the effective gene editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been widely used for knockout of genes in T. gondii. This review summarizes the applications of the CRISPR/Cas9 technology in vaccines against single- and double-gene deletion strains of T. gondii, so as to provide insights into development of toxoplasmosis vaccines.},
}

*CRISPR-Cas Systems
Humans
Animals
*Toxoplasma/genetics/immunology
*Toxoplasmosis/prevention & control/immunology/parasitology
Protozoan Vaccines/immunology
Gene Editing/methods

@article {pmid39622959,
year = {2024},
author = {Fang, B and Wang, C and Yuan, Y and Liu, X and Shi, L and Li, L and Wang, Y and Dai, Y and Yang, H},
title = {Generation and characterization of genetically modified pigs with GGTA1/β4GalNT2/CMAH knockout and human CD55/CD47 expression for xenotransfusion studies.},
journal = {Scientific reports},
volume = {14},
number = {1},
pages = {29870},
pmid = {39622959},
issn = {2045-2322},
support = {81970164//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Humans ; *Animals, Genetically Modified ; *Galactosyltransferases/genetics/metabolism ; Swine ; *CD55 Antigens/genetics/metabolism ; *CD47 Antigen/genetics/metabolism ; *Gene Knockout Techniques/methods ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Transplantation, Heterologous ; CRISPR-Cas Systems ; Erythrocytes/metabolism ; Macaca fascicularis/genetics ; Immunoglobulin G ; },
abstract = {Pig red blood cells (pRBCs) represent a promising alternative to address the shortage in transfusion medicine. Nonetheless, a major obstacle to their clinical implementation is immunological rejection. In this study, we generated transgenic pigs expressing human CD47 (hCD47) and CD55 (hCD55) in α1,3-galactosyltransferase KO/β-1,4-N-acetyl-galactosaminyl transferase 2 KO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO (TKO) pigs using CRISPR/Cas9 technology. Compared to wild-type pRBCs, TKO/hCD47/hCD55 pRBCs exhibit significantly reduced human IgG/IgM antibody binding. Moreover, when transfused into Cynomolgus monkeys, TKO/hCD47/hCD55 pRBCs remained detectable for 2 h post-transfusion, whereas wild-type pRBCs were eliminated within 20 min. This study demonstrates the potential of multi-gene edited pigs to provide immunologically compatible pRBCs.},
}

Animals
Humans
*Animals, Genetically Modified
*Galactosyltransferases/genetics/metabolism
Swine
*CD55 Antigens/genetics/metabolism
*CD47 Antigen/genetics/metabolism
*Gene Knockout Techniques/methods
*N-Acetylgalactosaminyltransferases/genetics/metabolism
*Mixed Function Oxygenases/genetics/metabolism
*Transplantation, Heterologous
CRISPR-Cas Systems
Erythrocytes/metabolism
Macaca fascicularis/genetics
Immunoglobulin G

@article {pmid39622625,
year = {2024},
author = {Mallozzi, A and Fusco, V and Ragazzini, F and di Bernardo, D},
title = {A Biomolecular Circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR Technology.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00225},
pmid = {39622625},
issn = {2161-5063},
abstract = {We introduce a biomolecular circuit for precise control of gene expression in mammalian cells. The circuit leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9 and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as robust perfect adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. This system belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of the CRISPR-Cas system. Finally, we show that RPA also holds in cells genomically integrated with the circuit, thus paving the way for practical applications in biotechnology that require stable cell lines.},
}

@article {pmid39621555,
year = {2024},
author = {Emaldi, M and Rey-Iborra, E and Marín, Á and Mosteiro, L and Lecumberri, D and Øyjord, T and Roncier, N and Mælandsmo, GM and Angulo, JC and Errarte, P and Larrinaga, G and Pulido, R and López, JI and Nunes-Xavier, CE},
title = {Impact of B7-H3 expression on metastasis, immune exhaustion and JAK/STAT and PI3K/AKT pathways in clear cell renal cell carcinoma.},
journal = {Oncoimmunology},
volume = {13},
number = {1},
pages = {2419686},
pmid = {39621555},
issn = {2162-402X},
mesh = {*Carcinoma, Renal Cell/pathology/immunology/genetics/metabolism/drug therapy ; Humans ; *B7 Antigens/metabolism/genetics ; *Kidney Neoplasms/pathology/immunology/metabolism/genetics/drug therapy ; *Proto-Oncogene Proteins c-akt/metabolism ; *Phosphatidylinositol 3-Kinases/metabolism ; *Signal Transduction/drug effects ; Janus Kinases/metabolism ; Cell Line, Tumor ; STAT Transcription Factors/metabolism ; Male ; Female ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Middle Aged ; Neoplasm Metastasis ; Aged ; Immune System Exhaustion ; },
abstract = {Immune checkpoint inhibitors in combination with tyrosine kinase inhibitors (TKIs) are improving the response rates of advanced renal cancer patients. However, many treated patients do not respond, making novel immune checkpoint-based immunotherapies potentially clinically beneficial only for specific groups of patients. We detected high expression of the immune checkpoint protein B7-H3 in clear cell renal cell carcinomas (ccRCCs) and evaluated B7-H3 immunohistochemistry staining in tissue microarray samples from two distinct renal cancer cohorts. B7-H3 was highly expressed in approximately 50% of primary tumors and in 30% of metastatic lesions. B7-H3 expression in primary tumors correlated with tumor necrosis, sarcomatoid transformation, disease-free survival, and synchronous metastasis, while B7-H3 expression in metastasis correlated with metastases to the lymph nodes. Gene expression analysis revealed the association of B7-H3 expression with gene expression scores involved in T cell exhaustion and myeloid immune evasion, as well as with PI3K/AKT and JAK/STAT pathways. Furthermore, knocking down B7-H3 expression in renal cancer cells by siRNA and CRISPR/Cas resulted in lower 2D and 3D cell proliferation and viability as well as increased sensitivity to TKI axitinib. Together, our findings suggest a pro-oncogenic and immune evasive role for B7-H3 in ccRCC and highlight B7-H3 as an actionable novel immune checkpoint protein in combination with TKI in advanced renal cancer.},
}

*Carcinoma, Renal Cell/pathology/immunology/genetics/metabolism/drug therapy
Humans
*B7 Antigens/metabolism/genetics
*Kidney Neoplasms/pathology/immunology/metabolism/genetics/drug therapy
*Proto-Oncogene Proteins c-akt/metabolism
*Phosphatidylinositol 3-Kinases/metabolism
*Signal Transduction/drug effects
Janus Kinases/metabolism
Cell Line, Tumor
STAT Transcription Factors/metabolism
Male
Female
Cell Proliferation
Gene Expression Regulation, Neoplastic
Middle Aged
Neoplasm Metastasis
Aged
Immune System Exhaustion

@article {pmid39529229,
year = {2024},
author = {Zhang, R and Ng, R and Wu, ST and Su, CY},
title = {Targeted deletion of olfactory receptors in D. melanogaster via CRISPR/Cas9-mediated LexA knock-in.},
journal = {Journal of neurogenetics},
volume = {38},
number = {3},
pages = {122-133},
doi = {10.1080/01677063.2024.2426014},
pmid = {39529229},
issn = {1563-5260},
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; *Receptors, Odorant/genetics ; *Gene Knock-In Techniques/methods ; *Olfactory Receptor Neurons/physiology/metabolism ; Drosophila Proteins/genetics ; Animals, Genetically Modified ; Gene Editing/methods ; },
abstract = {The study of olfaction in Drosophila melanogaster has greatly benefited from genetic reagents such as olfactory receptor mutant lines and GAL4 reporter lines. The CRISPR/Cas9 gene-editing system has been increasingly used to create null receptor mutants or replace coding regions with GAL4 reporters. To further expand this toolkit for manipulating fly olfactory receptor neurons (ORNs), we generated null alleles for 11 different olfactory receptors by using CRISPR/Cas9 to knock in LexA drivers, including multiple lines for receptors which have thus far lacked knock-in mutants. The targeted neuronal types represent a broad range of antennal ORNs from all four morphological sensillum classes. Additionally, we confirmed their loss-of-function phenotypes, assessed receptor haploinsufficiency, and evaluated the specificity of the LexA knock-in drivers. These receptor mutant lines have been deposited at the Bloomington Drosophila Stock Center for use by the broader scientific community.},
}

Animals
*Drosophila melanogaster/genetics
*CRISPR-Cas Systems
*Receptors, Odorant/genetics
*Gene Knock-In Techniques/methods
*Olfactory Receptor Neurons/physiology/metabolism
Drosophila Proteins/genetics
Animals, Genetically Modified
Gene Editing/methods

@article {pmid39392366,
year = {2024},
author = {Diao, LT and Xie, SJ and Xu, WY and Zhang, HH and Hou, YR and Hu, YX and Liang, XX and Liang, JB and Zhang, Q and Xiao, ZD},
title = {CRISPR/Cas13 sgRNA-Mediated RNA-RNA Interaction Mapping in Live Cells with APOBEC RNA Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {11},
number = {45},
pages = {e2409004},
doi = {10.1002/advs.202409004},
pmid = {39392366},
issn = {2198-3844},
support = {81974436//National Natural Science Foundation of China/ ; 82273158//National Natural Science Foundation of China/ ; 82472770//National Natural Science Foundation of China/ ; 2019ZT08Y485//Program for Guangdong Introducing Innovative and Entrepreneurial Teams/ ; 2022A1515010704//Natural Science Foundation of Guangdong/ ; 2023A03J0204//Science and Technology Projects in Guangzhou/ ; 2024A03J0176//Science and Technology Projects in Guangzhou/ ; 2024A04J6571//Science and Technology Projects in Guangzhou/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *RNA Editing/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; APOBEC Deaminases/metabolism/genetics ; RNA/genetics/metabolism ; },
abstract = {Current research on long non-coding RNA (lncRNA) has predominantly focused on identifying their protein partners and genomic binding sites, leaving their RNA partners largely unknown. To address this gap, the study has developed a method called sarID (sgRNA scaffold assisted RNA-RNA interaction detection), which integrates Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing to investigate lncRNA-RNA interactomes. By applying sarID to the lncRNA NEAT1, over one thousand previously unidentified binding transcripts are discovered. sarID is further expanded to investigate binders of XIST, MALAT1, NBR2, and DANCR, demonstrating its broad applicability in identifying lncRNA-RNA interactions. The findings suggest that lncRNAs may regulate gene expression by interacting with mRNAs, expanding their roles beyond known functions as protein scaffolds, miRNA sponges, or guides for epigenetic modulators. sarID has the potential to be adapted for studying other specific RNAs, providing a novel immunoprecipitation-free method for uncovering RNA partners and facilitating the exploration of the RNA-RNA interactome.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*RNA Editing/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
APOBEC Deaminases/metabolism/genetics
RNA/genetics/metabolism

@article {pmid39375921,
year = {2024},
author = {Lemak, S and Brown, G and Makarova, KS and Koonin, EV and Yakunin, AF},
title = {Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins.},
journal = {The FEBS journal},
volume = {291},
number = {23},
pages = {5177-5194},
doi = {10.1111/febs.17295},
pmid = {39375921},
issn = {1742-4658},
support = {//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; R-Loop Structures/genetics ; Protein Binding ; RNA, Bacterial/genetics/metabolism ; },
abstract = {The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.},
}

*Escherichia coli/genetics/metabolism
*Escherichia coli Proteins/metabolism/genetics/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
R-Loop Structures/genetics
Protein Binding
RNA, Bacterial/genetics/metabolism

@article {pmid39360620,
year = {2025},
author = {Wan, M and Zhao, D and Lin, S and Wang, P and Liang, B and Jin, Q and Jiao, Y and Song, Y and Ge, X and King, GJ and Yang, G and Wang, J and Hong, D},
title = {Allelic Variation of BnaFTA2 and BnaFTC6 Is Associated With Flowering Time and Seasonal Crop Type in Rapeseed (Brassica napus L.).},
journal = {Plant, cell & environment},
volume = {48},
number = {1},
pages = {852-865},
doi = {10.1111/pce.15165},
pmid = {39360620},
issn = {1365-3040},
support = {//This work was supported by the earmarked fund for CARS-12 and the Key Research & Development Programme of Hubei Province (2022BBA0038, 2022BBE001)./ ; },
mesh = {*Brassica napus/genetics/physiology/growth & development ; *Flowers/genetics/physiology/growth & development ; *Seasons ; *Alleles ; *Plant Proteins/genetics/metabolism ; Crops, Agricultural/genetics/physiology/growth & development ; Haplotypes/genetics ; Gene Expression Regulation, Plant ; Genetic Variation ; CRISPR-Cas Systems ; },
abstract = {Different ecological types of rapeseed (Brassica napus L.), including winter, spring, and semi-winter cultivars, exhibit varying flowering times and cannot be planted in the same cultivation areas. FLOWERING LOCUS T (FT) plays a key role in regulating flowering. In allotetraploid B. napus six copies of FT (BnaFT) have been reported. However, there is uncertainty about how the translated products of each paralog, as well as cis-allelic variations at each locus, contribute functionally to flowering time and define specific crop types. In this study, we confirm that BnaFT exhibit distinct expression patterns in different crop types of rapeseed. Using the CRISPR/Cas9 gene editing system, we provide functional evidence that the mutants between Bnaft paralogues affects the regulation of flowering time. Furthermore, we identify a new haplotype of BnaFT.A2 that is associated with early flowering time, although this appears necessary but not sufficient to confer a spring type phenotype. Three haplotypes of BnaFT.C6 were further identified and associated with both flowering time and crop types. We speculate that variations in both BnaFT.A2 and BnaFT.C6 may have undergone diversifying selection during the divergence of seasonal crop types in rapeseed.},
}

*Brassica napus/genetics/physiology/growth & development
*Flowers/genetics/physiology/growth & development
*Seasons
*Alleles
*Plant Proteins/genetics/metabolism
Crops, Agricultural/genetics/physiology/growth & development
Haplotypes/genetics
Gene Expression Regulation, Plant
Genetic Variation
CRISPR-Cas Systems

@article {pmid39621264,
year = {2025},
author = {Gowripalan, A and Smith, SA and Tscharke, DC},
title = {Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2860},
number = {},
pages = {115-130},
pmid = {39621264},
issn = {1940-6029},
mesh = {*Vaccinia virus/genetics ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Recombination, Genetic ; Poxviridae/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; Transfection/methods ; },
abstract = {Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient. Cas9-mediated selection is a technique that repurposes Cas9/guide RNA complexes to amplify chosen subsets of vaccinia viruses. We refer to this approach as Cas9-mediated poxvirus recombinant recovery (CASPRR). Transient introduction of appropriately guided Cas9 allows for recovery of marker-free recombinant viruses in just 5 days, and requires no additional virus modification. Following three rounds of purification, pure virus stocks are obtained. A newer method, stable expression of Cas9 and guide RNAs in a permissive cell line, allows for additional process streamlining, removing cell type-specific concerns related to transient transfection of Cas9. Within this chapter, the protocol for CASPRR is described in both a transient and stable expression model. These methods can be utilized to accelerate recovery of recombinant vaccinia viruses and be applied to generation of vaccinia libraries or novel therapeutic agents.},
}

*Vaccinia virus/genetics
Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Recombination, Genetic
Poxviridae/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Cell Line
Transfection/methods

@article {pmid39621263,
year = {2025},
author = {Lin, YJ and Evans, DH and Noyce, RS},
title = {Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2860},
number = {},
pages = {97-114},
pmid = {39621263},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Vaccinia virus/genetics ; *Poxviridae/genetics ; Humans ; Genome, Viral ; DNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Recombination, Genetic ; },
abstract = {The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Vaccinia virus/genetics
*Poxviridae/genetics
Humans
Genome, Viral
DNA, Viral/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Recombination, Genetic

@article {pmid39620587,
year = {2024},
author = {Cui, Z and Yuan, P and Zhao, Z and Zhao, F and Lu, L},
title = {Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {213},
pages = {},
doi = {10.3791/66966},
pmid = {39620587},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Th17 Cells/immunology/cytology ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Retroviridae/genetics ; Gene Targeting/methods ; Flow Cytometry/methods ; },
abstract = {T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay. Conventional Th17 polarization methods typically involve obtaining naïve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate naïve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.},
}

Animals
Mice
*Th17 Cells/immunology/cytology
*Cell Differentiation/genetics
*CRISPR-Cas Systems/genetics
*Retroviridae/genetics
Gene Targeting/methods
Flow Cytometry/methods

@article {pmid39563182,
year = {2024},
author = {Wang, Y and Chen, P and Wen, H and Gui, Y and Yan, D and Huang, D and Wang, D and Tang, BZ and Tan, H},
title = {Advanced Nanoplatform Mediated by CRISPR-Cas9 and Aggregation-Induced Emission Photosensitizers to Boost Cancer Theranostics.},
journal = {ACS nano},
volume = {18},
number = {48},
pages = {33168-33180},
doi = {10.1021/acsnano.4c11757},
pmid = {39563182},
issn = {1936-086X},
mesh = {*Photosensitizing Agents/chemistry/pharmacology ; *Theranostic Nanomedicine ; Humans ; *CRISPR-Cas Systems/genetics ; Mice ; Animals ; *Reactive Oxygen Species/metabolism ; Nanoparticles/chemistry ; Neoplasms/therapy/genetics/diagnostic imaging/pathology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/chemistry ; Immunotherapy ; Mice, Inbred BALB C ; Cell Proliferation/drug effects ; Drug Screening Assays, Antitumor ; },
abstract = {Immunotherapy combined with phototherapy is emerging as a promising strategy to treat omnipotent cancers. In this study, a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, aggregation-induced emission (AIE) photosensitizer (PS) and surface coating of polyethylene imine/hyaluronic acid were combined to construct a multifunctional nanoplatform, denoted as TCPH nanoparticles (NPs), for comprehensive cancer theranostics. TCPH NPs are featured by intrinsic functions including efficient reactive oxygen species (ROS) production, good photothermal conversion, programmed death-ligand 1 (PD-L1)-eliminating capability, and effective intracellular transport. The generated ROS and hyperthermia do not only achieve primary tumor elimination but also regulate the tumor immune microenvironment. Genomic disruption of PD-L1 conspicuously augments its therapeutic efficacy, especially in tumor metastasis and recurrence. Exceptional multimodal imaging navigation has also been developed. Excellent theranostics performance was substantiated in diverse tumor models, implying that this synergistic strategy of phototheranostics and immunotherapy provides a paradigm shift in emerging CRISPR-mediated nanomedicines.},
}

*Photosensitizing Agents/chemistry/pharmacology
*Theranostic Nanomedicine
Humans
*CRISPR-Cas Systems/genetics
Mice
Animals
*Reactive Oxygen Species/metabolism
Nanoparticles/chemistry
Neoplasms/therapy/genetics/diagnostic imaging/pathology
Cell Line, Tumor
Antineoplastic Agents/pharmacology/chemistry
Immunotherapy
Mice, Inbred BALB C
Cell Proliferation/drug effects
Drug Screening Assays, Antitumor

@article {pmid39561207,
year = {2024},
author = {Osia, B and Merkell, A and Lopezcolorado, FW and Ping, X and Stark, JM},
title = {RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.},
journal = {PLoS genetics},
volume = {20},
number = {11},
pages = {e1011479},
doi = {10.1371/journal.pgen.1011479},
pmid = {39561207},
issn = {1553-7404},
mesh = {*Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *Genomic Instability ; *Mitosis/genetics ; Humans ; *DNA Helicases/genetics/metabolism ; Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; DNA Repair/genetics ; Anaphase/genetics ; CRISPR-Cas Systems ; HeLa Cells ; Synthetic Lethal Mutations/genetics ; DNA Damage/genetics ; },
abstract = {Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis.},
}

*Rad52 DNA Repair and Recombination Protein/genetics/metabolism
*Genomic Instability
*Mitosis/genetics
Humans
*DNA Helicases/genetics/metabolism
Poly-ADP-Ribose Binding Proteins/genetics/metabolism
DNA Repair/genetics
Anaphase/genetics
CRISPR-Cas Systems
HeLa Cells
Synthetic Lethal Mutations/genetics
DNA Damage/genetics

@article {pmid39521232,
year = {2024},
author = {Yang, Q and Liu, J and Yu, Y and Cao, Y and Liu, C and Su, H and Huang, T and Liu, S and Yuan, J and Zhao, Z and Li, L and Cao, L and Gong, P},
title = {Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR/Cas12a technology.},
journal = {International journal of biological macromolecules},
volume = {282},
number = {Pt 6},
pages = {136912},
doi = {10.1016/j.ijbiomac.2024.136912},
pmid = {39521232},
issn = {1879-0003},
mesh = {Animals ; *Feces/parasitology ; *Fasciola hepatica/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Recombinases/metabolism/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Fascioliasis/diagnosis/parasitology/veterinary ; Sheep ; Sensitivity and Specificity ; },
abstract = {Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.},
}

Animals
*Feces/parasitology
*Fasciola hepatica/genetics/isolation & purification
*CRISPR-Cas Systems
*Recombinases/metabolism/genetics
Humans
Nucleic Acid Amplification Techniques/methods
Fascioliasis/diagnosis/parasitology/veterinary
Sheep
Sensitivity and Specificity

@article {pmid39490876,
year = {2024},
author = {Yang, Z and Li, H and Luo, M and Yi, H and Han, X and Liu, E and Yao, S and Hu, Z},
title = {Identification of c.146G > A mutation in a Fabry patient and its correction by customized Cas9 base editors in vitro.},
journal = {International journal of biological macromolecules},
volume = {282},
number = {Pt 3},
pages = {136922},
doi = {10.1016/j.ijbiomac.2024.136922},
pmid = {39490876},
issn = {1879-0003},
mesh = {*Fabry Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *alpha-Galactosidase/genetics/metabolism ; Female ; *CRISPR-Cas Systems ; Mutation ; Mutation, Missense ; },
abstract = {Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by mutations in the GLA gene, leading to reduced α-galactosidase (α-Gal A) activity. Current treatments, like enzyme replacement, have limitations affecting efficacy and patient outcomes. CRISPR/Cas9 genome editing tools may offer the potential to develop therapeutic strategy via correcting GLA mutations. In this study, we diagnosed a female FD patient with a missense mutation in exon 1 of the GLA gene (c.146G > A, p.R49H). Bioinformatic predictions and biochemical analyses in GLA-knockout cells revealed that this mutation significantly reduced α-Gal A stability and activity, confirming its pathogenicity. To correct this, we used adenine base editing. The mutation, along with a nearby bystander A, was efficiently edited by the traditional N-terminal adenine base editor. To avoid unwanted bystander editing, we developed a series of domain-inlaid base editors with the aim of narrowing editing window. The most effective variant, with deaminase inserted between the 947th and 948th residues of the RUVC3 domain, was further optimized by modifying linker rigidity. These adjustments shifted the editing window, eliminating bystander editing. Our findings clarify the pathogenic nature of a novel GLA mutation and demonstrate the potential of a customized base editor for therapeutic application in FD.},
}

*Fabry Disease/genetics/therapy
Humans
*Gene Editing/methods
*alpha-Galactosidase/genetics/metabolism
Female
*CRISPR-Cas Systems
Mutation
Mutation, Missense

@article {pmid39461690,
year = {2024},
author = {Yang, X and Zhou, S and Zeng, J and Zhang, S and Li, M and Yue, F and Chen, Z and Dong, Y and Zeng, Y and Luo, J},
title = {A biodegradable lipid nanoparticle delivers a Cas9 ribonucleoprotein for efficient and safe in situ genome editing in melanoma.},
journal = {Acta biomaterialia},
volume = {190},
number = {},
pages = {531-547},
doi = {10.1016/j.actbio.2024.10.030},
pmid = {39461690},
issn = {1878-7568},
mesh = {Animals ; *Gene Editing/methods ; *Nanoparticles/chemistry ; *Melanoma/pathology/therapy ; Mice ; *Lipids/chemistry ; *CRISPR-Associated Protein 9/metabolism ; Cell Line, Tumor ; *Proto-Oncogene Proteins B-raf/genetics ; Ribonucleoproteins/metabolism ; CRISPR-Cas Systems ; Humans ; Melanoma, Experimental/pathology/therapy ; Liposomes ; },
abstract = {The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.},
}

Animals
*Gene Editing/methods
*Nanoparticles/chemistry
*Melanoma/pathology/therapy
Mice
*Lipids/chemistry
*CRISPR-Associated Protein 9/metabolism
Cell Line, Tumor
*Proto-Oncogene Proteins B-raf/genetics
Ribonucleoproteins/metabolism
CRISPR-Cas Systems
Humans
Melanoma, Experimental/pathology/therapy
Liposomes

@article {pmid39448884,
year = {2024},
author = {Protasoni, M and López-Polo, V and Stephan-Otto Attolini, C and Brandariz, J and Herranz, N and Mateo, J and Ruiz, S and Fernandez-Capetillo, O and Kovatcheva, M and Serrano, M},
title = {Cyclophilin D plays a critical role in the survival of senescent cells.},
journal = {The EMBO journal},
volume = {43},
number = {23},
pages = {5972-6000},
pmid = {39448884},
issn = {1460-2075},
support = {HORIZON-MSCA-2021-PF-01//EC | Horizon Europe | Excellent Science | HORIZON EUROPE Marie Sklodowska-Curie Actions (MSCA)/ ; FPU-18/05917//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; PRYCO211023SERR//Fundación Científica Asociación Española Contra el Cáncer (AECC)/ ; CP19/00170//MEC | Instituto de Salud Carlos III (ISCIII)/ ; POSTD18020SERR//European Molecular Biology Organization (EMBO)/ ; PRYCO211023SERR//'la Caixa' Foundation ('la Caixa')/ ; Grup de Recerca consolidat 2017 SGR 282//Centres de Recerca de Catalunya (CERCA)/ ; },
mesh = {*Peptidyl-Prolyl Isomerase F/metabolism/genetics ; *Cellular Senescence ; Humans ; *Calcium/metabolism ; *Mitochondrial Permeability Transition Pore/metabolism ; *Mitochondria/metabolism ; *Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Cell Survival ; CRISPR-Cas Systems ; Cyclosporine/pharmacology ; Cyclophilins/metabolism/genetics ; Peptidyl-Prolyl Isomerase D ; },
abstract = {Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca[2+] and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca[2+] influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca[2+] ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells.},
}

*Peptidyl-Prolyl Isomerase F/metabolism/genetics
*Cellular Senescence
Humans
*Calcium/metabolism
*Mitochondrial Permeability Transition Pore/metabolism
*Mitochondria/metabolism
*Mitochondrial Membrane Transport Proteins/metabolism/genetics
Cell Survival
CRISPR-Cas Systems
Cyclosporine/pharmacology
Cyclophilins/metabolism/genetics
Peptidyl-Prolyl Isomerase D

@article {pmid39307305,
year = {2024},
author = {Liu, P and Jiang, J and Chen, Y and Gao, F and Wang, S and Yu, M and Liu, Y and Guo, R and Zhang, L and Xu, Z and Wang, C and Qi, X and Zhang, Y and Cui, H and Duan, Y and Wu, S and Gao, Y},
title = {Identification of Cables1 as a critical host factor that promotes ALV-J replication via genome-wide CRISPR/Cas9 gene knockout screening.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {11},
pages = {107804},
pmid = {39307305},
issn = {1083-351X},
mesh = {Animals ; *Virus Replication ; *Chickens ; *CRISPR-Cas Systems ; *Avian Leukosis Virus/genetics ; Gene Knockout Techniques ; Cell Line ; Ubiquitination ; Avian Leukosis/virology/metabolism/genetics ; Avian Proteins/genetics/metabolism ; },
abstract = {Avian leukosis virus subgroup J (ALV-J), a member of the genus Alpharetrovirus, possesses a small genome and exploits a vast array of host factors during its replication cycle. To identify host factors required for ALV-J replication and potentially guide the development of key therapeutic targets for ALV-J prevention, we employed a chicken genome-wide CRISPR/Cas9 knockout library to screen host factors involved in ALV-J infection within DF-1 cells. This screening revealed 42 host factors critical for ALV-J infection. Subsequent knockout assays showed that the absence of the genes encoding cycle-regulatory proteins, namely, Cables1, CDK1, and DHFR, significantly inhibited ALV-J replication. Notably, Cables1 knockout cell lines displayed the most pronounced inhibitory effect. Conversely, overexpression assays confirmed that Cables1 significantly promotes ALV-J replication. Immunoprecipitation assays further indicated that Cables1 specifically interacts with the viral protein p15 (viral protease) among all ALV-J proteins, enhancing ALV-J p15 polyubiquitination. Additionally, we identified 26 lysine residues of ALV-J p15 as key sites for ubiquitination, and their replacement with arginine attenuated the replication ability of ALV-J in both in vitro and in vivo assays. This study demonstrates that Cables1 is a critical replication-dependent host factor of ALV-J by enhancing p15 ubiquitination and thereby promoting viral replication. Overall, these findings contribute to a deeper understanding of the ALJ-V replication mechanism and offer a potential target for the prevention and control of ALV-J infection.},
}

Animals
*Virus Replication
*Chickens
*CRISPR-Cas Systems
*Avian Leukosis Virus/genetics
Gene Knockout Techniques
Cell Line
Ubiquitination
Avian Leukosis/virology/metabolism/genetics
Avian Proteins/genetics/metabolism

@article {pmid39305958,
year = {2024},
author = {Jo, M and Lee, JS and Tocheny, CE and Lero, MW and Bui, QT and Morgan, JS and Shaw, LM},
title = {Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function.},
journal = {The Journal of biological chemistry},
volume = {300},
number = {11},
pages = {107796},
pmid = {39305958},
issn = {1083-351X},
support = {R01 CA227993/CA/NCI NIH HHS/United States ; R01 CA229910/CA/NCI NIH HHS/United States ; },
mesh = {*Insulin Receptor Substrate Proteins/metabolism/genetics ; Humans ; *Indoleacetic Acids/metabolism ; Proteolysis ; Cell Nucleus/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; Degrons ; },
abstract = {Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.},
}

*Insulin Receptor Substrate Proteins/metabolism/genetics
Humans
*Indoleacetic Acids/metabolism
Proteolysis
Cell Nucleus/metabolism
HEK293 Cells
CRISPR-Cas Systems
Degrons

@article {pmid38901107,
year = {2024},
author = {Jiang, L and Wang, P and Li, C and Shen, D and Chen, A and Qian, H and Zhao, Q},
title = {Compensatory effects of other olfactory genes after CRISPR/cas9 editing of BmOR56 in silkworm, Bombyx mori.},
journal = {Comparative biochemistry and physiology. Part D, Genomics & proteomics},
volume = {52},
number = {},
pages = {101275},
doi = {10.1016/j.cbd.2024.101275},
pmid = {38901107},
issn = {1878-0407},
mesh = {*Bombyx/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Gene Editing ; Receptors, Odorant/genetics/metabolism ; Morus/genetics ; },
abstract = {Bombyx mori is an oligophagous economic insect. Cis-Jasmone is one of the main substances in mulberry leaf that attract silkworm for feeding and BmOR56 is its receptor. Potential interaction ways between BmOR56 and cis-Jasmone were explored, which included some crucial amino acids such as Gln172, Val173, Ser176, Lys182, His322, and Arg345. BmOR56 was edited using CRISPR/cas9 for Qiufeng, and a homozygous knockout strain QiufengM was obtained. Compared with Qiufeng, the feeding ability of QiufengM on mulberry leaf did not change significantly, but on artificial diet decreased significantly. QiufengM also showed a dependence on the concentration of mulberry leaf powder. The result indicated that other olfactory genes had a compensatory effect on the attractance of mulberry leaf after the loss of BmOR56. Transcriptome analysis of antennae showed that many genes differentially expressed between Qiufeng and QiufengM, which involved in olfactory system, glucose metabolism, protein metabolism, amino acid metabolism, and insect hormone biosynthesis. Particularly, BmIR21, BmOR53 and BmOR27 were significantly up-regulated, which may have a compensatory effect on BmOR56 loss. In addition, detoxification mechanism was activated and may cause the passivation of feeling external signals in silkworm.},
}

*Bombyx/genetics/metabolism
Animals
*CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
*Gene Editing
Receptors, Odorant/genetics/metabolism
Morus/genetics

@article {pmid39619730,
year = {2024},
author = {Ahmed, MM and Kayode, HH and Okesanya, OJ and Ukoaka, BM and Eshun, G and Mourid, MR and Adigun, OA and Ogaya, JB and Mohamed, ZO and Lucero-Prisno, DE},
title = {CRISPR-Cas Systems in the Fight Against Antimicrobial Resistance: Current Status, Potentials, and Future Directions.},
journal = {Infection and drug resistance},
volume = {17},
number = {},
pages = {5229-5245},
pmid = {39619730},
issn = {1178-6973},
abstract = {BACKGROUND: Antimicrobial resistance (AMR) is a critical global health concern that threatens the efficacy of existing antibiotics and poses significant challenges to public health and the economy worldwide. This review explores the potential of CRISPR-Cas systems as a novel approach to combating AMR and examines current applications, limitations, and prospects.

METHODS: A comprehensive literature search was conducted across multiple databases, including PubMed, Google Scholar, Scopus, and Web of Science, covering publications published from 2014 to August 2024. This review focuses on CRISPR-Cas technologies and their applications in AMR.

RESULTS: CRISPR-Cas systems have demonstrated efficacy in combating antimicrobial resistance by targeting and eliminating antibiotic-resistance genes. For example, studies have shown that CRISPR-Cas9 can effectively target and eliminate colistin resistance genes in MCR-1 plasmids, restoring susceptibility to carbapenems in bacteria such as E. coli and Klebsiella pneumoniae. Further molecular findings highlight the impact of CRISPR-Cas systems on various bacterial species, such as Enterococcus faecalis, in which CRISPR systems play a crucial role in preventing the acquisition of resistance genes. The effectiveness of CRISPR-Cas in targeting these genes varies due to differences in CRISPR locus formation among bacterial species. For instance, variations in CRISPR loci influence the targeting of resistance genes in E. faecalis, and CRISPR-Cas9 successfully reduces resistance by targeting genes such as tetM and ermB.

CONCLUSION: CRISPR-Cas systems are promising for fighting AMR by targeting and eliminating antibiotic-resistant genes, as demonstrated by the effective targeting of colistin resistance genes on MCR-1 plasmids and their similar activities. However, the effectiveness of CRISPR-Cas is affected by variations in the CRISPR loci among bacterial species. Challenges persist, such as optimizing delivery methods and addressing off-target effects to ensure the safety and precision of CRISPR-Cas systems in clinical settings.},
}

@article {pmid39617792,
year = {2024},
author = {Hosseinpour, M and Xi, X and Liu, L and Malaver-Ortega, L and Perlaza-Jimenez, L and Joo, JE and York, HM and Beesley, J and Caldon, CE and Dugué, PA and Dowty, JG and Arumugam, S and Southey, MC and Rosenbluh, J},
title = {SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {10449},
pmid = {39617792},
issn = {2041-1723},
support = {2011329//Department of Health | National Health and Medical Research Council (NHMRC)/ ; MCRF20035//Victorian Cancer Agency (VCA)/ ; },
mesh = {*DNA Methylation ; Humans ; *Breast Neoplasms/genetics/metabolism/drug therapy ; *DNA Methyltransferase 3A/metabolism ; Female ; *DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Receptors, Estrogen/metabolism/genetics ; MCF-7 Cells ; Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {DNA methylation is an epigenetic mark that plays a critical role in regulating gene expression. DNA methyltransferase (DNMT) inhibitors, inhibit global DNA methylation and have been a key tool in studies of DNA methylation. A major bottleneck is the lack of tools to induce global DNA methylation. Here, we engineered a CRISPR based approach, that we initially designed, to enable site-specific DNA methylation. Using the synergistic activation mediator (SAM) system, we unexpectedly find that regardless of the targeted sequence any sgRNA induces global genome-wide DNA methylation. We term this method SAM-DNMT3A and show that induction of global DNA methylation is a unique vulnerability in ER-positive breast cancer suggesting a therapeutic approach. Our findings highlight the need of caution when using CRISPR based approaches for inducing DNA methylation and demonstrate a method for global induction of DNA methylation.},
}

*DNA Methylation
Humans
*Breast Neoplasms/genetics/metabolism/drug therapy
*DNA Methyltransferase 3A/metabolism
Female
*DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Receptors, Estrogen/metabolism/genetics
MCF-7 Cells
Epigenesis, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39617755,
year = {2024},
author = {Wu, Y and Huang, Z and Liu, Y and He, P and Wang, Y and Yan, L and Wang, X and Gao, S and Zhou, X and Yoon, CW and Sun, K and Situ, Y and Ho, P and Zeng, Y and Yuan, Z and Zhu, L and Zhou, Q and Zhao, Y and Liu, T and Kwong, GA and Chien, S and Liu, L and Wang, Y},
title = {Ultrasound Control of Genomic Regulatory Toolboxes for Cancer Immunotherapy.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {10444},
pmid = {39617755},
issn = {2041-1723},
support = {EB029122//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; GM140929//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; HL121365//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; HD107206//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; CA262815//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; EB032822//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; K01EB035649//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Neoplasms/therapy/genetics/immunology ; Animals ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; Dependovirus/genetics ; Immunotherapy/methods ; Genomics/methods ; Ultrasonic Waves ; },
abstract = {There remains a critical need for the precise control of CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies. Here, we engineer a set of inducible CRISPR-based tools controllable by focused ultrasound (FUS), which can penetrate deep and induce localized hyperthermia for transgene activation. We demonstrate the capabilities of FUS-inducible CRISPR, CRISPR activation (CRISPRa), and CRISPR epigenetic editor (CRISPRee) in modulating the genome and epigenome. We show that FUS-CRISPR-mediated telomere disruption primes solid tumours for chimeric antigen receptor (CAR)-T cell therapy. We further deliver FUS-CRISPR in vivo using adeno-associated viruses (AAVs), followed by FUS-induced telomere disruption and the expression of a clinically validated antigen in a subpopulation of tumour cells, functioning as "training centers" to activate synthetic Notch (synNotch) CAR-T cells to produce CARs against a universal tumour antigen to exterminate neighboring tumour cells. The FUS-CRISPR(a/ee) toolbox hence allows the noninvasive and spatiotemporal control of genomic/epigenomic reprogramming for cancer treatment.},
}

Humans
*Neoplasms/therapy/genetics/immunology
Animals
*CRISPR-Cas Systems
*Receptors, Chimeric Antigen/metabolism/genetics
Mice
Cell Line, Tumor
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats
Immunotherapy, Adoptive/methods
Dependovirus/genetics
Immunotherapy/methods
Genomics/methods
Ultrasonic Waves

@article {pmid39616957,
year = {2024},
author = {Fu, Z and Zhao, L and Guo, Y and Yang, J},
title = {Gene therapy for hereditary hearing loss.},
journal = {Hearing research},
volume = {455},
number = {},
pages = {109151},
doi = {10.1016/j.heares.2024.109151},
pmid = {39616957},
issn = {1878-5891},
abstract = {Gene therapy is a technique by which exogenous genetic material is introduced into target cells to treat or prevent diseases caused by genetic mutations. Hearing loss is the most common sensory disorder. Genetic factors contribute to approximately 50 % of all cases of profound hearing loss, and more than 150 independent genes have been reported as associated with hearing loss. Recent advances in CRISPR/Cas based gene-editing tools have facilitated the development of gene therapies for hereditary hearing loss (HHL). Viral delivery vectors, and especially adeno-associated virus (AAV) vectors, have been demonstrated as safe and efficient carriers for the delivery of transgenes into inner ear cells in animal models. More importantly, AAV-mediated gene therapy can restore hearing in some children with hereditary deafness. However, there are many different types of HHL that need to be identified and evaluated to determine appropriate gene therapy options. In the present review, we summarize recent animal model-based advances in gene therapy for HHL, as well as gene therapy strategies, gene-editing tools, delivery vectors, and administration routes. We also discuss the strengths and limitations of different gene therapy methods and describe future challenges for the eventual clinical application of gene therapy for HHL.},
}

@article {pmid39616917,
year = {2024},
author = {Nassereddine, ZN and Opara, SD and Coutinho, OA and Qyteti, F and Book, R and Heinicke, MP and Napieralski, J and Tiquia-Arashiro, SM},
title = {Critical perspectives on advancing antibiotic resistant gene (ARG) detection technologies in aquatic ecosystems.},
journal = {The Science of the total environment},
volume = {957},
number = {},
pages = {177775},
doi = {10.1016/j.scitotenv.2024.177775},
pmid = {39616917},
issn = {1879-1026},
abstract = {The spread of antibiotic resistance genes (ARGs) in aquatic ecosystems poses a serious risk to environmental and public health, making advanced detection and monitoring methods essential. This review provides a fresh perspective and a critical evaluation of recent advances in detecting and monitoring ARGs in aquatic environments. It highlights the latest innovations in molecular, bioinformatic, and environmental techniques. While traditional methods like culture-based assays and polymerase chain reaction (PCR) remain important, they are increasingly being supplemented by high-throughput sequencing technologies applied to metagenomics. These technologies offer comprehensive insights into the diversity and distribution of ARGs in aquatic environments. The integration of bioinformatic tools and databases has improved the accuracy and efficiency of ARG detection, enabling the analysis of complex datasets and tracking the evolution of ARGs in aquatic settings. Additionally, new environmental monitoring methods, including novel biosensors, geographic information systems (GIS) applications, and remote sensing technologies, have emerged as powerful tools for real-time ARG surveillance in water systems. This review critically examines the challenges of standardizing these methodologies and emphasizes the need for interdisciplinary approaches to enhance ARG monitoring across different aquatic ecosystems. By assessing the strengths and limitations of various methods, this review aims to guide future research and the development of more effective strategies for managing antibiotic resistance in aquatic environments.},
}

@article {pmid39616576,
year = {2025},
author = {Ng, EFY and Meitinger, F},
title = {Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2872},
number = {},
pages = {167-187},
pmid = {39616576},
issn = {1940-6029},
mesh = {Humans ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Genetic Testing/methods ; },
abstract = {Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.},
}

Humans
*Gene Editing/methods
*Gene Knockout Techniques/methods
*CRISPR-Cas Systems
Genetic Engineering/methods
Genetic Testing/methods

@article {pmid39616566,
year = {2025},
author = {Hatano, Y and Yonezawa, N and Tokoro, M and Yao, T and Yamagata, K},
title = {Chromosome Counting at Meiosis and Mitosis of Mouse Oocytes and Embryos Using Super-Resolution Live-Cell Imaging and CRISPR/dCas9-Mediated Live-FISH.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2872},
number = {},
pages = {21-35},
pmid = {39616566},
issn = {1940-6029},
mesh = {Animals ; Mice ; *Oocytes/cytology/metabolism ; *Meiosis/genetics ; *In Situ Hybridization, Fluorescence/methods ; *CRISPR-Cas Systems ; *Mitosis/genetics ; Female ; Embryo, Mammalian/cytology ; Microscopy, Confocal/methods ; Chromosome Segregation ; Chromosomes, Mammalian/genetics ; },
abstract = {Live cell imaging techniques are now essential for capturing chromosomal segregation in fertilized eggs. Although better spatiotemporal resolution of fluorescence observations could provide more information, higher phototoxicity may occur. Super-resolution microscopy is generally considered unsuitable for live cell imaging because of the considerable cell damage. Here, we developed a method for counting chromosomes in mouse living oocytes and early embryos using super-resolution microscopy based on disk confocal photon reassignment microscopy (OPRA). In this chapter, we describe the imaging conditions for minimally invasive, high-resolution observation of mouse oocytes and embryos and a method to count chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization.},
}

Animals
Mice
*Oocytes/cytology/metabolism
*Meiosis/genetics
*In Situ Hybridization, Fluorescence/methods
*CRISPR-Cas Systems
*Mitosis/genetics
Female
Embryo, Mammalian/cytology
Microscopy, Confocal/methods
Chromosome Segregation
Chromosomes, Mammalian/genetics

@article {pmid39615910,
year = {2025},
author = {Amouzadeh Tabrizi, M and Ali, AA and Singuru, MMR and Mi, L and Bhattacharyya, P and You, M},
title = {A portable and sensitive DNA-based electrochemical sensor for detecting piconewton-scale cellular forces.},
journal = {Analytica chimica acta},
volume = {1333},
number = {},
pages = {343392},
pmid = {39615910},
issn = {1873-4324},
support = {R35 GM133507/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Electrochemical Techniques/instrumentation/methods ; *DNA/chemistry/analysis ; HeLa Cells ; *Biosensing Techniques/instrumentation ; Smartphone ; Electrodes ; Gold/chemistry ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. The measurement of forces exerted and experienced by cells is fundamental in understanding these mechanosensitive cellular behaviors. While cell-generated forces can now be detected based on techniques like fluorescence microscopy, atomic force microscopy, optical/magnetic tweezers, however, most of these approaches rely on complicated instruments or materials, as well as skilled operators, which could limit their potential broad applications in regular biological laboratories.

RESULTS: A new type of smartphone-based electrochemical sensor is developed here for cellular force measurement. In this system, a double-stranded DNA-based force probe, known as tension gauge tether, is attached to the surface of a gold screen-printed electrode, which is then incorporated into a portable smartphone-based electrochemical device. Cellular force-induced DNA detachment on the sensor surface results in multiple redox reporters to reach the surface of the electrode and generate enhanced electrochemical signals. To further improve the sensitivity, a CRISPR-Cas12a system has also been incorporated to cleave the remaining surface-attached anchor DNA strand. Using integrin-mediated tension as an example, piconewton-scale adhesion forces generated by ≤ 10 HeLa cells could now be reliably detected. Meanwhile, the threshold forces of these electrochemical sensors can also be modularly tuned to detect different levels of cellular forces.

SIGNIFICANCE: These novel DNA-based highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be potentially powerful tools for detecting different cell-generated molecular forces. Functioning as complementary tools with traction force microscopy and fluorescent probes, these electrochemical sensors can be straightforwardly applied in regular biological laboratories for understanding the basic mechanical principles of cell signaling and for developing novel strategies and materials in tissue engineering, regenerative medicine, and cell therapy.},
}

Humans
*Electrochemical Techniques/instrumentation/methods
*DNA/chemistry/analysis
HeLa Cells
*Biosensing Techniques/instrumentation
Smartphone
Electrodes
Gold/chemistry
CRISPR-Cas Systems

@article {pmid39615707,
year = {2024},
author = {Kadkhoda, H and Gholizadeh, P and Ghotaslou, R and Nabizadeh, E and Pirzadeh, T and Ahangarzadeh Rezaee, M and Feizi, H and Samadi Kafil, H and Aghazadeh, M},
title = {Role of CRISPR-Cas System on Virulence traits and Carbapenem Resistance in Clinical Klebsiella pneumoniae Isolates.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107151},
doi = {10.1016/j.micpath.2024.107151},
pmid = {39615707},
issn = {1096-1208},
abstract = {BACKGROUND AND OBJECTIVE(S): The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence.

MATERIALS AND METHODS: A total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR.

RESULTS: The development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW.

CONCLUSION: CRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.},
}

@article {pmid39614269,
year = {2024},
author = {Wang, Y and Yu, F and Zhang, K and Shi, K and Chen, Y and Li, J and Li, X and Zhang, L},
title = {End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific.},
journal = {BMC veterinary research},
volume = {20},
number = {1},
pages = {540},
pmid = {39614269},
issn = {1746-6148},
support = {22A310019//Key Scientific Research Foundation of the Higher Education Institutions of Henan Province/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Special Project of Henan Province of China/ ; },
mesh = {*Enterocytozoon/genetics/isolation & purification ; *Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Microsporidiosis/veterinary/diagnosis/microbiology ; Animals ; DNA, Fungal/genetics ; Limit of Detection ; Feces/microbiology ; Dogs ; },
abstract = {Enterocytozoon bieneusi is a common species of microsporidia that infects humans and animals. Current methods for detecting E. bieneusi infections have trade-offs in sensitivity, specificity, simplicity, cost and speed and are thus unacceptable for clinical application. We tested the effectiveness of a previously reported CRISPR/Cas12a-based method (ReCTC) when used for the nucleic acid detection of E. bieneusi. The limit of detection (LOD) and the specificity of the expanded ReCTC were evaluated using prepared target DNA, and the accuracy of the ReCTC-based detection of E. bieneusi in clinical samples was validated. The ReCTC method was successfully used for the nucleic acid detection of E. bieneusi. The sensitivity test indicated an LOD of 3.7 copies/µl for the ReCTC-based fluorescence and lateral flow strip methods. In specificity test involving other common enteric pathogens, a fluorescent signal and/or test line appeared only when the sample was positive for E. bieneusi. These results demonstrated that the ReCTC method can successfully detect E. bieneusi in clinical samples. The ReCTC method was successfully used to detect E. bieneusi nucleic acid with high sensitivity and specificity. It had excellent performance in clinical DNA samples and was superior to nested polymerase chain reaction. Furthermore, the ReCTC method demonstrated its capability for use in on-site detection.},
}

*Enterocytozoon/genetics/isolation & purification
*Sensitivity and Specificity
*CRISPR-Cas Systems
*Microsporidiosis/veterinary/diagnosis/microbiology
Animals
DNA, Fungal/genetics
Limit of Detection
Feces/microbiology
Dogs

@article {pmid39614172,
year = {2024},
author = {Yang, W and Zhu, JK and Jin, W},
title = {A catalog of gene editing sites and genetic variations in editing sites in model organisms.},
journal = {BMC genomics},
volume = {25},
number = {1},
pages = {1153},
pmid = {39614172},
issn = {1471-2164},
support = {2021YFA1300404//National Key R&D Program of China/ ; 2021YFA0909300//National Key R&D Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 32170646//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing ; Animals ; Humans ; *CRISPR-Cas Systems ; *Genetic Variation ; Mice ; Receptors, CCR5/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Oryza/genetics ; Polymorphism, Single Nucleotide ; Arabidopsis/genetics ; },
abstract = {BACKGROUND: CRISPR-Cas systems require a protospacer adjacent motif (PAM), which plays an essential role in self/non-self discrimination in their natural context, to cleave DNA for genome editing. Unfortunately, common genetic variation is distributed throughout genomes, which can block recognition of target sites by Cas proteins. However, little information is available about the distribution of editing sites in model organisms and how often common variation overlaps with those PAM sites.

RESULTS: Herein, we characterized six representative Cas proteins (Cas9, Cas12a, Cas12b, Cas12i, Cas12j and Cas12l) genomic editing sites in ten model organisms (yeast, flatworms, flies, zebrafish, mice, humans, rice, maize, Arabidopsis and tomato). We demonstrated that there were more than 34 editing sites per kilobase on average in these genomes. In each genome, 91.69-99.83% and 95.4-99.73% of genes had at least one unique editing site in exon and promoter, respectively. Depending on publicly available genomic diversity data, we identified the variations (SNPs and InDels) in editing sites in humans and rice, indicating the risk in the application of CRISPR/Cas technology. Finally, using CCR5 and BCL11A as examples, we revealed variation site was a factor that must be considered when designing sgRNA.

CONCLUSIONS: Our findings not only revealed the distribution characteristics of editing sites of six representative Cas proteins in ten model organism genomes but also shed light on the adverse effect of variation sites on target site recognition. Our current work will serve as a reminder of the risks of CRISPR application.},
}

*Gene Editing
Animals
Humans
*CRISPR-Cas Systems
*Genetic Variation
Mice
Receptors, CCR5/genetics
CRISPR-Associated Proteins/genetics/metabolism
Oryza/genetics
Polymorphism, Single Nucleotide
Arabidopsis/genetics

@article {pmid39521064,
year = {2024},
author = {Guzman Gonzalez, V and Grunenberger, A and Nicoud, O and Czuba, E and Vollaire, J and Josserand, V and Le Guével, X and Desai, N and Coll, JL and Divita, G and Faure, V},
title = {Enhanced CRISPR-Cas9 RNA system delivery using cell penetrating peptides-based nanoparticles for efficient in vitro and in vivo applications.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {376},
number = {},
pages = {1160-1175},
doi = {10.1016/j.jconrel.2024.11.008},
pmid = {39521064},
issn = {1873-4995},
mesh = {Animals ; *CRISPR-Cas Systems ; *Cell-Penetrating Peptides/chemistry ; *Nanoparticles/chemistry ; Humans ; Gene Transfer Techniques ; Gene Editing/methods ; Mice ; RNA/administration & dosage ; Female ; Cell Line, Tumor ; Mice, Inbred BALB C ; },
abstract = {CRISPR-Cas9 system has emerged as a revolutionary gene-editing tool with huge therapeutic potential for addressing the underlying genetic causes of various diseases, including cancer. However, there are challenges such as the delivery method that must be overcome for its clinical application. In addition to the risk of nuclease degradation and rapid clearance of the CRISPR-Cas9 system by macrophages, the large size of Cas9, the high anionic charge density and hydrophilic nature of the RNA hinder their intracellular delivery and overall gene transfection efficiency. In this study, we engineered a novel Peptide-Based Nanoparticles ADGN for the delivery of long RNA. ADGN peptides can form stable self-assembled nanoparticles with CRISPR-Cas9 RNA. They have the ability to cross the cell membrane of various cell types, exhibiting a preference for cancer cells that overexpress laminin receptor and safeguard RNA prior their delivery into the cytoplasm. We demonstrate that ADGN peptides significantly promote CRISPR-Cas9 mediated knockout of the luciferase gene in vitro achieving 60 % efficiency with a preference for G insertion at the targeted site of luciferase gene. Moreover, we have provided evidence that these nanoparticles can also be systemically intravenously administrated in vivo in mice to deliver a functional CRISPR-Cas9 system to tumoral lung cells orthotopically implanted in the mouse, resulting in an effective gene knockout in mice. We also demonstrated that the in vivo distribution of ADGN-RNA is influenced by its peptides to RNA molar ratio. This study introduces a promising new Peptide-Based Nanoparticles for delivering CRISPR-Cas9 system in its RNA form applicable in both in vitro and in vivo models.},
}

Animals
*CRISPR-Cas Systems
*Cell-Penetrating Peptides/chemistry
*Nanoparticles/chemistry
Humans
Gene Transfer Techniques
Gene Editing/methods
Mice
RNA/administration & dosage
Female
Cell Line, Tumor
Mice, Inbred BALB C

@article {pmid39520794,
year = {2024},
author = {Tripathi, JN and Muiruri, S and Tripathi, L},
title = {Advancements and challenges in gene editing for improvement of vegetatively propagated crops.},
journal = {Current opinion in plant biology},
volume = {82},
number = {},
pages = {102653},
doi = {10.1016/j.pbi.2024.102653},
pmid = {39520794},
issn = {1879-0356},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; },
abstract = {Gene editing technologies, particularly CRISPR-Cas9, have revolutionized agriculture by offering precise and efficient tools to enhance crop production. The vegetatively propagated crops, crucial for global food security, face challenges such as climate change, pests, and limited genetic diversity. CRISPR-Cas9 enables targeted modifications to improve traits like disease resistance, drought tolerance, and nutritional content, thereby boosting productivity and sustainability. Despite its transformative potential, the adoption of gene editing in vegetatively propagated crops is hampered by technical complexities and regulatory frameworks. This review explores recent advancements, challenges, and prospects of gene editing in vegetatively propagated crops, emphasizing strategies to overcome technical barriers and regulatory constraints. Addressing these issues is essential for realizing the full agricultural potential of gene editing and ensuring food security in a changing global climate.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics
*CRISPR-Cas Systems

@article {pmid39504623,
year = {2024},
author = {Wang, Y and Ma, J and Wu, Y and Yang, S and Wang, P and Zhang, H and Li, J and Chen, L and Kong, W and Xia, Y and Wang, Q and Liu, J},
title = {A simple, cost-effective, and efficient method for screening CRISPR/Cas9 mutants in plants.},
journal = {Journal of plant physiology},
volume = {303},
number = {},
pages = {154375},
doi = {10.1016/j.jplph.2024.154375},
pmid = {39504623},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems ; *Mutation ; *Gene Editing/methods ; Cost-Benefit Analysis ; Polymerase Chain Reaction/methods ; Plants/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-Bsl I-associated analysis (PCR-BAA). This method requires only standard PCR and Bsl I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.},
}

*CRISPR-Cas Systems
*Mutation
*Gene Editing/methods
Cost-Benefit Analysis
Polymerase Chain Reaction/methods
Plants/genetics

@article {pmid39488143,
year = {2025},
author = {Lynch, CRH and Martin, OL and Billington, C and Fleming, R},
title = {Towards the identification of body fluids using RT-LAMP isothermal amplification coupled with CRISPR-Cas12a.},
journal = {Forensic science international. Genetics},
volume = {74},
number = {},
pages = {103167},
doi = {10.1016/j.fsigen.2024.103167},
pmid = {39488143},
issn = {1878-0326},
mesh = {Humans ; *Nucleic Acid Amplification Techniques/methods ; Female ; Male ; *Menstruation ; *Semen/chemistry ; *Cervix Mucus/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; Genetic Markers ; Mucins/genetics ; Rectum/metabolism ; Vagina/metabolism ; Body Fluids/chemistry ; Forensic Genetics/methods ; Molecular Diagnostic Techniques ; },
abstract = {While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative technique that enables detection of mRNA at a single temperature (usually 60-65℃) for 10-30 minutes and has comparable sensitivity to PCR. We describe the coupling of RT-LAMP amplification (60℃ for 30 minutes) with CRISPR-mediated fluorescent detection of the body fluid specific mRNA markers MMP3 (menstrual fluid), CYP2B7P (vaginal material), TNP1 (spermatozoa), KLK2 (semen), and MUC12 (rectal mucosa). Following temperature optimization and final selection of RT-LAMP-CRISPR assays, their specificity across circulatory blood, buccal, menstrual fluid, vaginal material, semen, and rectal mucosa was assessed. Most assays were specific for their intended target body fluid, although MMP3 and CYP2B7P were detected in some rectal mucosa samples, the latter of which has been observed previously in the literature. A preliminary sensitivity assessment in target fluids was determined by a dilution series over six logs of RNA input. A range of assay approaches were investigated to develop a protocol suitable for use in a forensic screening laboratory. This included the determination of fluorescent assay results by eye, use of lyophilised reagents, and RT-LAMP and CRISPR reactions undertaken in one-tube in a lower resource setting.},
}

Humans
*Nucleic Acid Amplification Techniques/methods
Female
Male
*Menstruation
*Semen/chemistry
*Cervix Mucus/chemistry
RNA, Messenger/genetics
CRISPR-Cas Systems
Genetic Markers
Mucins/genetics
Rectum/metabolism
Vagina/metabolism
Body Fluids/chemistry
Forensic Genetics/methods
Molecular Diagnostic Techniques

@article {pmid39481850,
year = {2024},
author = {Soares De Oliveira, L and Kaserman, JE and Van Der Spek, AH and Lee, NJ and Undeutsch, HJ and Werder, RB and Wilson, AA and Hollenberg, AN},
title = {Thyroid hormone receptor beta (THRβ1) is the major regulator of T3 action in human iPSC-derived hepatocytes.},
journal = {Molecular metabolism},
volume = {90},
number = {},
pages = {102057},
doi = {10.1016/j.molmet.2024.102057},
pmid = {39481850},
issn = {2212-8778},
mesh = {Humans ; *Hepatocytes/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Thyroid Hormone Receptors beta/metabolism/genetics ; *Cell Differentiation ; *Triiodothyronine/metabolism/pharmacology ; Signal Transduction ; Cells, Cultured ; Cell Proliferation ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: Thyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THRβ1 is likely the main isoform expressed in liver, its role in human hepatocytes is not fully understood.

METHODS: To elucidate the role of THRβ1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THRβ1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage, iPSC-derived hepatocytes were then interrogated to determine the role of THRβ1 in ligand-independent and -dependent functions.

RESULTS: We found that the loss of THRβ1 promoted alterations in proliferation rate and metabolic pathways regulated by T3, including gluconeogenesis, lipid oxidation, fatty acid synthesis, and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THRβ1 signaling mechanisms. Finally, we demonstrate that following THRβ1 knockout, several key metabolic genes remain T3 responsive suggesting they are THRα targets.

CONCLUSIONS: These results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes.},
}

Humans
*Hepatocytes/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*Thyroid Hormone Receptors beta/metabolism/genetics
*Cell Differentiation
*Triiodothyronine/metabolism/pharmacology
Signal Transduction
Cells, Cultured
Cell Proliferation
CRISPR-Cas Systems

@article {pmid39394696,
year = {2024},
author = {Koga, D and Nakayama, S and Higa, T and Nakayama, KI},
title = {Identification and characterization of an enhancer element regulating expression of Cdkn1c (p57 gene).},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {29},
number = {12},
pages = {1264-1274},
doi = {10.1111/gtc.13173},
pmid = {39394696},
issn = {1365-2443},
support = {KAKENHI grants (JP23H00378)//Japan Society for the Promotion of Science/ ; KAKENHI grants (JP23K14197)//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Cyclin-Dependent Kinase Inhibitor p57/genetics/metabolism ; Mice ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; KCNQ1 Potassium Channel/genetics/metabolism ; Embryonic Development/genetics ; },
abstract = {The mammalian p57 protein is a member of the CIP/KIP family of cyclin-dependent kinase inhibitors and plays an essential role in the development of multiple tissues during embryogenesis as well as in the maintenance of tissue stem cells in adults. Although several transcription factors have been implicated in regulating the p57 gene, cis-elements such as enhancers that regulate its expression have remained ill-defined. Here we identify a candidate enhancer for the mouse p57 gene (Cdkn1c) within an intron of the Kcnq1 locus by 4C-seq analysis in mouse embryonic stem cells (mESCs). Deletion of this putative enhancer region with the CRISPR-Cas9 system or its suppression by CRISPR interference resulted in a marked attenuation of Cdkn1c expression in differentiating mESCs. Our results thus suggest that this region may serve as an enhancer for the p57 gene during early mouse embryogenesis.},
}

Animals
*Cyclin-Dependent Kinase Inhibitor p57/genetics/metabolism
Mice
*Enhancer Elements, Genetic
*Mouse Embryonic Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Cell Differentiation/genetics
KCNQ1 Potassium Channel/genetics/metabolism
Embryonic Development/genetics

@article {pmid39322759,
year = {2024},
author = {Matsuura-Suzuki, E and Kiyokawa, K and Iwasaki, S and Tomari, Y},
title = {miRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms.},
journal = {The EMBO journal},
volume = {43},
number = {23},
pages = {6161-6179},
pmid = {39322759},
issn = {1460-2075},
support = {18H05271//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05278//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21K06026//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Pioneering Project//MEXT | RIKEN ()/ ; Aging Project//MEXT | RIKEN ()/ ; },
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics ; *Larva/growth & development/metabolism/genetics ; *Drosophila melanogaster/growth & development/genetics/metabolism ; *MicroRNAs/genetics/metabolism ; Gene Silencing ; Argonaute Proteins/metabolism/genetics ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.},
}

Animals
*Drosophila Proteins/metabolism/genetics
*Larva/growth & development/metabolism/genetics
*Drosophila melanogaster/growth & development/genetics/metabolism
*MicroRNAs/genetics/metabolism
Gene Silencing
Argonaute Proteins/metabolism/genetics
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
RNA-Binding Proteins/metabolism/genetics

@article {pmid39278589,
year = {2024},
author = {Robertson, NR and Trivedi, V and Lupish, B and Ramesh, A and Aguilar, Y and Carrera, S and Lee, S and Arteaga, A and Nguyen, A and Lenert-Mondou, C and Harland-Dunaway, M and Jinkerson, R and Wheeldon, I},
title = {Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica.},
journal = {Metabolic engineering},
volume = {86},
number = {},
pages = {55-65},
doi = {10.1016/j.ymben.2024.09.005},
pmid = {39278589},
issn = {1096-7184},
mesh = {*Yarrowia/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering ; Genome, Fungal ; },
abstract = {CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of ∼50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.},
}

*Yarrowia/genetics/metabolism
*CRISPR-Cas Systems
*Metabolic Engineering
Genome, Fungal

@article {pmid39242074,
year = {2024},
author = {Shin, SW and Min, H and Kim, J and Lee, JS},
title = {A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations.},
journal = {Metabolic engineering},
volume = {86},
number = {},
pages = {12-28},
doi = {10.1016/j.ymben.2024.09.004},
pmid = {39242074},
issn = {1096-7184},
mesh = {*Doxycycline/pharmacology ; Humans ; *Gain of Function Mutation ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Engineering/methods ; Cell Line ; Animals ; },
abstract = {For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.},
}

*Doxycycline/pharmacology
Humans
*Gain of Function Mutation
CRISPR-Cas Systems
HEK293 Cells
Cell Engineering/methods
Cell Line
Animals

@article {pmid39190085,
year = {2024},
author = {Li, T and Li, W and Li, F and Lin, J and Zhang, Y and Zhang, Q and Sun, Y and Chen, X and Zhou, S and Li, Q},
title = {Effects of two chd2-knockout strains on the morphology and behavior in zebrafish.},
journal = {Development genes and evolution},
volume = {234},
number = {2},
pages = {173-180},
pmid = {39190085},
issn = {1432-041X},
support = {NSFC, No. 81771632 and No. 81271509//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Behavior, Animal ; DNA-Binding Proteins/genetics/metabolism ; Embryo, Nonmammalian/metabolism ; },
abstract = {The chromodomain helicase DNA binding domain 2 (CHD2) gene is an ATPase and a member of the SNF2-like family of helicase-related enzymes. CHD2 plays critical roles in human brain development and function, and homozygous mutation of Chd2 in mice results in perinatal lethality. To further elucidate the effects of chd2, we used CRISPR/Cas9 to create two chd2-knockout strains (fdu901, 11,979-11982delGGGT, and fdu902, 27350delG) in zebrafish. We found that the deformity and mortality rates of fdu901 and fdu902 were higher than those of the wild type. Developmental delay was more obvious and embryo mortality was higher in fdu901 than in fdu902. However, the embryo deformity rate in fdu902 was higher than that in fdu901. Although there were no significant differences in behavior between the two knockout zebrafish and wild-type zebrafish at 7 days post fertilization (dpf), fdu901 and fdu902 zebrafish showed different alterations. The excitability of fdu902 was higher than that of fdu901. Overall, our data demonstrate that two homozygous chd2 knockout mutations were survivable and could be stably inherited and that fdu901 and fdu902 zebrafish differed in behavior and morphology. These two models might be good tools for understanding the functions of the different domains of chd2.},
}

Animals
*Zebrafish/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Behavior, Animal
DNA-Binding Proteins/genetics/metabolism
Embryo, Nonmammalian/metabolism

@article {pmid39186687,
year = {2024},
author = {MacLeod, G and Molaei, F and Haider, S and Almeida, MP and Lin, S and Kushida, M and Sureshkumar, H and Bhatti, JK and Lu, JQ and Schramek, D and Dirks, PB and Angers, S},
title = {Fitness Screens Map State-Specific Glioblastoma Stem Cell Vulnerabilities.},
journal = {Cancer research},
volume = {84},
number = {23},
pages = {3967-3983},
doi = {10.1158/0008-5472.CAN-23-4024},
pmid = {39186687},
issn = {1538-7445},
support = {PJT-169054//Canadian Institutes of Health Research (CIHR)/ ; },
mesh = {*Glioblastoma/pathology/genetics/metabolism ; Humans ; *Neoplastic Stem Cells/pathology/metabolism ; *Brain Neoplasms/pathology/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Gene Expression Regulation, Neoplastic ; Mice ; Tumor Cells, Cultured ; Cell Line, Tumor ; },
abstract = {Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults and is driven by self-renewing glioblastoma stem cells (GSC) that persist after therapy and seed treatment-refractory recurrent tumors. GBM tumors display a high degree of intra- and intertumoral heterogeneity that is a prominent barrier to targeted treatment strategies. This heterogeneity extends to GSCs that exist on a gradient between two transcriptional states or subtypes termed developmental and injury response. Drug targets for each subtype are needed to effectively target GBM. To identify conserved and subtype-specific genetic dependencies across a large and heterogeneous panel of GSCs, we designed the GBM5K-targeted guide RNA library and performed fitness screens in a total of 30 patient-derived GSC cultures. The focused CRISPR screens identified the most conserved subtype-specific vulnerabilities in GSCs and elucidated the functional dependency gradient existing between the developmental and injury response states. Developmental-specific fitness genes were enriched for transcriptional regulators of neurodevelopment, whereas injury response-specific fitness genes were highlighted by several genes implicated in integrin and focal adhesion signaling. These context-specific vulnerabilities conferred differential sensitivity to inhibitors of β1 integrin, focal adhesion kinase, MEK, and OLIG2. Interestingly, the screens revealed that the subtype-specific signaling pathways drive differential cyclin D (CCND1 vs. CCND2) dependencies between subtypes. These data provide a biological insight and mechanistic understanding of GBM heterogeneity and point to opportunities for precision targeting of defined GBM and GSC subtypes to tackle heterogeneity. Significance: CRISPR-Cas9 screens in a panel of patient-derived glioblastoma stem cells reveal heterogeneity in genetic vulnerabilities across subtypes that have important implications for targeted and combination treatment strategies for glioblastoma.},
}

*Glioblastoma/pathology/genetics/metabolism
Humans
*Neoplastic Stem Cells/pathology/metabolism
*Brain Neoplasms/pathology/genetics/metabolism
CRISPR-Cas Systems
Animals
Gene Expression Regulation, Neoplastic
Mice
Tumor Cells, Cultured
Cell Line, Tumor

@article {pmid39003620,
year = {2024},
author = {VanWinkle, PE and Lee, E and Wynn, B and Nawara, TJ and Thomas, H and Parant, J and Alvarez, C and Serra, R and Sztul, E},
title = {Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {253},
number = {12},
pages = {1106-1129},
doi = {10.1002/dvdy.726},
pmid = {39003620},
issn = {1097-0177},
support = {U54 OD030167/OD/NIH HHS/United States ; 5T90DE022736-09/DE/NIDCR NIH HHS/United States ; R01GM122802/GM/NIGMS NIH HHS/United States ; R01GM122802/GM/NIGMS NIH HHS/United States ; 5T90DE022736-09/DE/NIDCR NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Animal Fins/physiology ; *Zebrafish Proteins/genetics/metabolism ; Regeneration/genetics/physiology ; Cyclic AMP Response Element-Binding Protein/genetics/metabolism ; Osteogenesis/genetics/physiology ; Body Patterning/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood.

RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration.

CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.},
}

Animals
*Zebrafish/genetics
*Animal Fins/physiology
*Zebrafish Proteins/genetics/metabolism
Regeneration/genetics/physiology
Cyclic AMP Response Element-Binding Protein/genetics/metabolism
Osteogenesis/genetics/physiology
Body Patterning/genetics
CRISPR-Cas Systems

@article {pmid38988258,
year = {2024},
author = {Georgiadis, C and Nickolay, L and Syed, F and Zhan, H and Gkazi, SA and Etuk, A and Abramowski-Mock, U and Preece, R and Cuber, P and Adams, S and Ottaviano, G and Qasim, W},
title = {Umbilical cord blood T cells can be isolated and enriched by CD62L selection for use in 'off the shelf' chimeric antigen receptor T-cell therapies to widen transplant options.},
journal = {Haematologica},
volume = {109},
number = {12},
pages = {3941-3951},
doi = {10.3324/haematol.2024.285101},
pmid = {38988258},
issn = {1592-8721},
mesh = {Humans ; *Receptors, Chimeric Antigen/immunology/genetics ; *Fetal Blood/cytology ; Animals ; Mice ; *T-Lymphocytes/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; Gene Editing ; Graft vs Host Disease/prevention & control/immunology ; },
abstract = {Umbilical cord blood (UCB) T cells exhibit distinct naïve ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR- Cas9 editing, T-cell expansion, and depletion of residual TCRαβ T cells. This provided stringent mitigation against the risk of graft-versus-host disease (GvHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCRαβ T cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterization captured vector integration sites and CRISPR editing signatures, and functional studies, including in vivo potency studies in humanized mice, confirmed anti-leukemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB-derived T-cell banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.},
}

Humans
*Receptors, Chimeric Antigen/immunology/genetics
*Fetal Blood/cytology
Animals
Mice
*T-Lymphocytes/immunology/metabolism
*Immunotherapy, Adoptive/methods
CRISPR-Cas Systems
Gene Editing
Graft vs Host Disease/prevention & control/immunology

@article {pmid38934080,
year = {2024},
author = {Kasprzyk, ME and Sura, W and Podralska, M and Kazimierska, M and Seitz, A and Łosiewski, W and Woźniak, T and Guikema, JEJ and Diepstra, A and Kluiver, J and Van den Berg, A and Rozwadowska, N and Dzikiewicz-Krawczyk, A},
title = {Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.},
journal = {Haematologica},
volume = {109},
number = {12},
pages = {4007-4020},
doi = {10.3324/haematol.2023.284672},
pmid = {38934080},
issn = {1592-8721},
mesh = {Humans ; *Enhancer Elements, Genetic ; *Lymphoma, Non-Hodgkin/genetics/pathology/mortality ; *Immunoglobulin Heavy Chains/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Cell Survival/genetics ; Translocation, Genetic ; },
abstract = {Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNA (eRNA). We designed a single guide RNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin- enriched RNA sequencing we showed transcription from the core enhancer regions and subsequently validated expression of the eRNA in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNA and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.},
}

Humans
*Enhancer Elements, Genetic
*Lymphoma, Non-Hodgkin/genetics/pathology/mortality
*Immunoglobulin Heavy Chains/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Cell Survival/genetics
Translocation, Genetic

@article {pmid38841800,
year = {2024},
author = {Davies, N and Francis, T and Oldreive, C and Azam, M and Wilson, J and Byrd, PJ and Burley, M and Sharma-Oates, A and Keane, P and Alatawi, S and Higgs, MR and Rudzki, Z and Ibrahim, M and Perry, T and Agathanggelou, A and Hewitt, AM and Smith, E and Bonifer, C and O'Connor, M and Forment, JV and Murray, PG and Fennell, E and Kelly, G and Chang, C and Stewart, GS and Stankovic, T and Kwok, M and Taylor, AM},
title = {Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.},
journal = {Haematologica},
volume = {109},
number = {12},
pages = {3989-4006},
doi = {10.3324/haematol.2023.284404},
pmid = {38841800},
issn = {1592-8721},
mesh = {*Lymphoma, Large B-Cell, Diffuse/genetics/metabolism/pathology ; Mice ; Animals ; Humans ; *Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; Nucleotides/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Knockout ; Disease Models, Animal ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied RNA sequencing and genome-scale loss-of-function clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/- lymphomas, which phenotypically resemble either activated B-cell-like or germinal center B-cell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/- lymphomas, we discovered nucleotide biosynthesis as a MYC-dependent cellular vulnerability that can be targeted through the synergistic nucleotide-depleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/- lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.},
}

*Lymphoma, Large B-Cell, Diffuse/genetics/metabolism/pathology
Mice
Animals
Humans
*Ataxia Telangiectasia Mutated Proteins/genetics/metabolism
Nucleotides/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Mice, Knockout
Disease Models, Animal
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid39614043,
year = {2025},
author = {Lei, T and Zhang, G},
title = {Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2874},
number = {},
pages = {9-20},
pmid = {39614043},
issn = {1940-6029},
mesh = {*Cdc20 Proteins/genetics/metabolism ; *Mitosis/genetics ; *RNA Interference ; *CRISPR-Cas Systems ; Humans ; Cell Line ; Kinetochores/metabolism ; M Phase Cell Cycle Checkpoints/genetics ; },
abstract = {Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.},
}

*Cdc20 Proteins/genetics/metabolism
*Mitosis/genetics
*RNA Interference
*CRISPR-Cas Systems
Humans
Cell Line
Kinetochores/metabolism
M Phase Cell Cycle Checkpoints/genetics

@article {pmid39612312,
year = {2024},
author = {Liu, Y and Liu, Q and Yi, C and Liu, C and Shi, Q and Wang, M and Han, F},
title = {Past innovations and future possibilities in plant chromosome engineering.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14530},
pmid = {39612312},
issn = {1467-7652},
support = {31991212//National Natural Science Foundation of China/ ; 2023ZD04025//Biological Breeding-National Science and Technology Major Project/ ; },
abstract = {Plant chromosome engineering has emerged as a pivotal tool in modern plant breeding, facilitating the transfer of desirable traits through the incorporation of alien chromosome fragments into plants. Here, we provide a comprehensive overview of the past achievements, current methodologies and future prospects of plant chromosome engineering. We begin by examining the successful integration of specific examples such as the incorporation of rye chromosome segments (e.g. the 1BL/1RS translocation), Dasypyrum villosum segments (e.g. the 6VS segment for powdery mildew resistance), Thinopyrum intermedium segments (e.g. rust resistance genes) and Thinopyrum elongatum segments (e.g. Fusarium head blight resistance genes). In addition to trait transfer, advancements in plant centromere engineering have opened new possibilities for chromosomal manipulation. This includes the development of plant minichromosomes via centromere-mediated techniques, the generation of haploids through CENH3 gene editing, and the induction of aneuploidy using KaryoCreate. The advent of CRISPR/Cas technology has further revolutionized chromosome engineering, enabling large-scale chromosomal rearrangements, such as inversions and translocations, as well as enabling targeted insertion of large DNA fragments and increasing genetic recombination frequency. These advancements have significantly expanded the toolkit for genetic improvement in plants, opening new horizons for the future of plant breeding.},
}

@article {pmid39611587,
year = {2024},
author = {Sayid, R and van den Hurk, AWM and Rothschild-Rodriguez, D and Herrema, H and de Jonge, PA and Nobrega, FL},
title = {Characteristics of phage-plasmids and their impact on microbial communities.},
journal = {Essays in biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1042/EBC20240014},
pmid = {39611587},
issn = {1744-1358},
support = {AUF 5188//Amsterdam University Fund/ ; PhD fellowship//Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers (ACS)/ ; //Bowel Research UK (BRUK)/ ; Aspasia premium (015.017.050)//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ ; BB/T008768/1//Biotechnology and Biological Sciences Research Council (BBSRC)/ ; Black Futures Research Studentship//University of Southampton (University of Southampton UK)/ ; },
abstract = {Bacteria host various foreign genetic elements, most notably plasmids and bacteriophages (or phages). Historically, these two classes were seen as separate, but recent research has shown considerable interplay between them. Phage-plasmids (P-Ps) exhibit characteristics of both phages and plasmids, allowing them to exist extrachromosomally within bacterial hosts as plasmids, but also to infect and lyse bacteria as phages. This dual functionality enables P-Ps to utilize the modes of transmission of both phage and plasmids, facilitating the rapid dissemination of genetic material, including antibiotic resistance and virulence genes, throughout bacterial populations. Additionally, P-Ps have been found to encode toxin-antitoxin and CRISPR-Cas adaptive immune systems, which enhance bacterial survival under stress and provide immunity against other foreign genetic elements. Despite a growing body of literature on P-Ps, large gaps remain in our understanding of their ecological roles and environmental prevalence. This review aims to synthesise existing knowledge and identify research gaps on the impacts of P-Ps on microbial communities.},
}

@article {pmid39610939,
year = {2024},
author = {Chu, LL and Sohng, JK and Bae, H and Dhakal, D},
title = {Editorial: Recent advances in application of synthetic biology for production of bioactive compounds, volume II.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {12},
number = {},
pages = {1517610},
doi = {10.3389/fbioe.2024.1517610},
pmid = {39610939},
issn = {2296-4185},
}

@article {pmid39563028,
year = {2024},
author = {Nieland, L and Vrijmoet, AB and Jetten, IW and Rufino-Ramos, D and de Reus, AJEM and Breyne, K and Kleinstiver, BP and Maguire, CA and Broekman, MLD and Breakefield, XO and Abels, ER},
title = {CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2024.11.023},
pmid = {39563028},
issn = {1525-0024},
abstract = {Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice.},
}

@article {pmid39414147,
year = {2025},
author = {Sohail, A},
title = {Methyltransferase 1 (OsMTS1) interacts with hydroxycinnamoyltransferase 1 (OsHCT1) and promotes heading by upregulating heading date 1 (Hd1).},
journal = {Plant science : an international journal of experimental plant biology},
volume = {350},
number = {},
pages = {112291},
doi = {10.1016/j.plantsci.2024.112291},
pmid = {39414147},
issn = {1873-2259},
mesh = {*Oryza/genetics/metabolism/enzymology ; *Plant Proteins/genetics/metabolism ; *Methyltransferases/genetics/metabolism ; *Plants, Genetically Modified/genetics ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Up-Regulation ; },
abstract = {Heading date determines the distribution and yield potentials of rice, and is an ideal target for crop improvement using CRISPR/Cas9 genome editing system. In this study, we reported the loss-of-function of Methyltransferase 1 (MTS1), which promotes heading in rice. Here, we constructed knockouts and overexpression transgenic plants of OsMTS1 in ZH8015 and Nipponbare (NIP) for the first time to validate its heading date function in rice subspecies Oryza sativa ssp. Indica and O. Sativa ssp. Japonica, respectively. The OsMTS1 knockouts in ZH8015 and NIP rice significantly promoted heading date under both natural short days (NSD) and natural long days (NLD) conditions, while the overexpression of OsMTS1 significantly delayed heading date in ZH8015 and NIP rice under both NSD and NLD conditions. Likewise, the complementation transgenic plants displayed late heading date phenotype. OsMTS1 repressed heading through up-regulating Heading date 1 (Hd1) and down-regulating Early heading date 1 (Ehd1) and Heading date 3a (Hd3a). The OsMTS1 protein interacted with OsHCT1 proteins using a yeast two-hybrid (Y2H) assay. The Y2H and overexpression confirmed that OsMTS1 interacted with OsHCT1, which delayed heading by 4.7 days under NLD. Taken together, CRISPR/Cas9, genetic complementation, and overexpression results validated that OsMTS1 represses heading in Indica and Japonica rice under both NLD and NSD conditions. These results demonstrated that OsMTS1 is a useful target for breeding early maturing rice varieties by CRISPR/Cas9 gene editing of the functional allele.},
}

*Oryza/genetics/metabolism/enzymology
*Plant Proteins/genetics/metabolism
*Methyltransferases/genetics/metabolism
*Plants, Genetically Modified/genetics
*Gene Expression Regulation, Plant
CRISPR-Cas Systems
Up-Regulation

@article {pmid39609592,
year = {2024},
author = {Geuverink, WP and Houtman, D and Retel Helmrich, IRA and Kist, JD and Henneman, L and Cornel, MC and Riedijk, SR and , },
title = {A decade of public engagement regarding human germline gene editing: a systematic scoping review.},
journal = {European journal of human genetics : EJHG},
volume = {},
number = {},
pages = {},
pmid = {39609592},
issn = {1476-5438},
abstract = {Following the discovery of the CRISPR-Cas technology in 2012, there has been a growing global call for public engagement regarding the potential use of human germline gene editing (HGGE). In this systematic scoping review, we aim to evaluate public engagement studies considering the following questions based on three points of attention: 1) Inclusion of underrepresented groups: who have been engaged? 2) Gathering values: what output has been reported? 3) Reaching societal impact: what objectives of public engagement have been reported? A systematic literature search from 2012 to 2023 identified 3464 articles reporting on public engagement studies regarding HGGE retrieved from 12 databases. After screening, 52 full-text articles were assessed for eligibility, resulting in 36 articles that cover 31 public engagement studies. We conclude that co-created efforts are needed to engage underrepresented groups as well as to yield values rather than acceptance levels, and to concretise how engagement might result in societal impact.},
}

@article {pmid39609418,
year = {2024},
author = {Miskalis, A and Shirguppe, S and Winter, J and Elias, G and Swami, D and Nambiar, A and Stilger, M and Woods, WS and Gosstola, N and Gapinske, M and Zeballos, A and Moore, H and Maslov, S and Gaj, T and Perez-Pinera, P},
title = {SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {10354},
pmid = {39609418},
issn = {2041-1723},
support = {1U01NS122102//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1U01NS122102//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01GM141296//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 1R01GM127497//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 1R01GM131272//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 1R01GM141296//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; 17SDG33650087//American Heart Association (American Heart Association, Inc.)/ ; },
mesh = {*Exons/genetics ; Animals ; *Gene Editing/methods ; Humans ; Mice ; *Alzheimer Disease/genetics/therapy/metabolism ; Disease Models, Animal ; HEK293 Cells ; CRISPR-Cas Systems ; Mice, Transgenic ; RNA Splice Sites/genetics ; Amyloid beta-Peptides/metabolism/genetics ; RNA Splicing ; },
abstract = {Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which have broad applications in medicine and biotechnology. Existing techniques including antisense oligonucleotides, targetable nucleases, and base editors, while effective for specific applications, remain hindered by transient effects, genotoxicity, and inconsistent exon skipping. To overcome these limitations, here we develop SPLICER, a toolbox of next-generation base editors containing near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing improves exon skipping, reduces aberrant splicing, and enables skipping of exons refractory to single splice site editing. To demonstrate the therapeutic potential of SPLICER, we target APP exon 17, which encodes amino acids that are cleaved to form Aβ plaques in Alzheimer's disease. SPLICER reduces the formation of Aβ42 peptides in vitro and enables efficient exon skipping in a mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and efficient exon skipping toolbox.},
}

*Exons/genetics
Animals
*Gene Editing/methods
Humans
Mice
*Alzheimer Disease/genetics/therapy/metabolism
Disease Models, Animal
HEK293 Cells
CRISPR-Cas Systems
Mice, Transgenic
RNA Splice Sites/genetics
Amyloid beta-Peptides/metabolism/genetics
RNA Splicing

@article {pmid39609269,
year = {2024},
author = {Li, C and Xiao, Y and Zhou, J and Liu, S and Zhang, L and Song, X and Guo, X and Song, Q and Zhao, J and Deng, N},
title = {Knockout of onecut2 inhibits proliferation and promotes apoptosis of tumor cells through SKP2-mediated p53 acetylation in hepatocellular carcinoma.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {81},
number = {1},
pages = {469},
pmid = {39609269},
issn = {1420-9071},
support = {81972705//National Natural Science Foundation of China/ ; },
mesh = {*S-Phase Kinase-Associated Proteins/metabolism/genetics ; *Carcinoma, Hepatocellular/pathology/metabolism/genetics ; Humans ; *Liver Neoplasms/pathology/metabolism/genetics ; *Apoptosis/genetics ; *Cell Proliferation/genetics ; Acetylation ; Animals ; *Tumor Suppressor Protein p53/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {Onecut2 (OC2) plays a vital regulatory role in tumor growth, metastasis and angiogenesis. In this study, we report the regulatory role and specific molecular mechanism of OC2 in the apoptosis of hepatocellular carcinoma (HCC) cells. We found that OC2 knockout via the CRISPR/CAS9 system not only significantly inhibited the proliferation and angiogenesis of HCC cells but also significantly promoted apoptosis. The apoptosis rate of the OC2 knockout HCC cell line reached 30.514%. In a mouse model, the proliferation inhibition rate of tumor cells reached 98.8%. To explore the mechanism of apoptosis, ChIP-Seq and dual-luciferase reporter assays were carried out. The results showed that OC2 could directly bind to the promotor of SKP2 and regulate its expression. Moreover, downregulating the expression of OC2 and SKP2 could release p300, promote the acetylation of p53, increase the expression of p21 and p27, and promote the apoptosis of HCC cells. Moreover, the overexpression of OC2 or SKP2 in the knockout HCC cell line clearly inhibited the acetylation level of p53 and reduced cell apoptosis. This study revealed that OC2 could regulate the apoptosis of HCC cells through the SKP2/p53/p21 axis, which may provide some therapeutic targets for HCC in the clinic.},
}

*S-Phase Kinase-Associated Proteins/metabolism/genetics
*Carcinoma, Hepatocellular/pathology/metabolism/genetics
Humans
*Liver Neoplasms/pathology/metabolism/genetics
*Apoptosis/genetics
*Cell Proliferation/genetics
Acetylation
Animals
*Tumor Suppressor Protein p53/metabolism/genetics
Mice
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Mice, Nude
Gene Knockout Techniques
CRISPR-Cas Systems/genetics

@article {pmid39566123,
year = {2024},
author = {Dara, M and Dianatpour, M and Azarpira, N and Tanideh, N and Tanideh, R},
title = {Integrating CRISPR technology with exosomes: Revolutionizing gene delivery systems.},
journal = {Biochemical and biophysical research communications},
volume = {740},
number = {},
pages = {151002},
doi = {10.1016/j.bbrc.2024.151002},
pmid = {39566123},
issn = {1090-2104},
mesh = {*Exosomes/genetics ; Humans ; *Gene Transfer Techniques ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Therapy/methods ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) serves as an adaptive immune system in bacteria and archaea, offering a defense mechanism against invading genetic elements such as viruses (bacteriophages) and plasmids. Today, CRISPR has evolved into a powerful gene-editing technology that enables highly specific and rapid modifications of DNA within a genome. It has a broad range of applications across various fields, including medicine, agriculture, and fundamental research. One of the significant challenges facing this technology is the efficient transfer of CRISPR constructs into target cells for gene editing. There are several methods to deliver this system into target cells, which can be classified as viral and non-viral methods. Each of these approaches has its own advantages and disadvantages. Recently, the use of extracellular vesicles for delivery has garnered particular attention. Exosomes are nano-sized extracellular vesicles that have emerged as promising carriers for drug delivery due to their unique properties. These naturally occurring vesicles, typically ranging from 30 to 150 nm in diameter, facilitate intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids between cells. Exosome therapy has surfaced as a promising strategy in regenerative medicine, utilizing small extracellular vesicles to deliver therapeutic molecules to target cells. One of the emerging options for transferring the CRISPR system is exosomes. The integration of these two advanced technologies holds significant potential for developing efficient and targeted gene editing and advancing precision medicine. In contemporary medicine, there is an increasing focus on personalized and targeted treatments that cater to the distinct genetic and molecular profiles of individual patients. The synergy of CRISPR technology and exosome therapy presents a remarkable opportunity to develop highly targeted and effective therapeutic strategies customized to individual patient requirements. This review article examines the potential of incorporating CRISPR technology within exosomes for precision therapeutic applications.},
}

*Exosomes/genetics
Humans
*Gene Transfer Techniques
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genetic Therapy/methods

@article {pmid39541214,
year = {2024},
author = {Guo, R and Sun, X and Wang, F and Han, D and Yang, Q and Gao, H and Li, Z and Shao, Z and Shi, J and Yang, R and Huo, X and Yan, J and Li, G and Xiao, Q and Liu, Y and Zhang, S and Liu, X and Zhou, Y and Wang, L and Hu, C and Xu, C},
title = {Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice.},
journal = {Cell reports},
volume = {43},
number = {11},
pages = {114973},
doi = {10.1016/j.celrep.2024.114973},
pmid = {39541214},
issn = {2211-1247},
mesh = {Animals ; *Dependovirus/genetics ; *Gene Editing/methods ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; Tyrosinemias/therapy/genetics ; Gene Transfer Techniques ; Disease Models, Animal ; Mice, Inbred C57BL ; HEK293 Cells ; Genetic Therapy/methods ; },
abstract = {IscBs, as hypercompact ancestry proteins of Cas9 nuclease, are suitable for in vivo gene editing via single adeno-associated virus (AAV) delivery. Due to the low activity of natural IscBs in eukaryotic cells, recent studies have been focusing on improving OgeuIscB's gene editing efficiency via protein engineering. However, in vivo gene editing efficacy of IscBs for disease correction remained to be demonstrated. Here, we showed effective gene knockout and base editing in mouse embryos. To further improve IscB activity, we performed systematic engineering of IscB-associated ωRNA and identified a variant, ωRNA[∗]-v2, with enhanced gene editing efficiency. Furthermore, our study demonstrated the efficacy of an engineered IscB-ωRNA system for robust gene knockout and base editing in vivo. Single AAV delivery of IscB-derived cytosine and adenine base editors achieved disease correction in a mouse model of tyrosinemia. Therefore, our results indicated the great potential of miniature IscBs for developing single-AAV-based gene editing therapeutics.},
}

Animals
*Dependovirus/genetics
*Gene Editing/methods
Mice
Humans
CRISPR-Cas Systems/genetics
Tyrosinemias/therapy/genetics
Gene Transfer Techniques
Disease Models, Animal
Mice, Inbred C57BL
HEK293 Cells
Genetic Therapy/methods

@article {pmid39541213,
year = {2024},
author = {Rahman, ASMZ and Syroegin, EA and Novomisky Nechcoff, J and Devarajan, A and Polikanov, YS and Cardona, ST},
title = {Rationally designed pooled CRISPRi-seq uncovers an inhibitor of bacterial peptidyl-tRNA hydrolase.},
journal = {Cell reports},
volume = {43},
number = {11},
pages = {114967},
doi = {10.1016/j.celrep.2024.114967},
pmid = {39541213},
issn = {2211-1247},
mesh = {*Carboxylic Ester Hydrolases/metabolism/genetics ; Burkholderia cenocepacia/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Library ; Bacterial Proteins/metabolism/genetics ; Enzyme Inhibitors/pharmacology ; Genes, Essential ; Mutation ; },
abstract = {Bacterial mutant libraries with downregulated antibiotic targets are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi). By modeling depletion levels and adjusting knockdown mutant inocula, we rationally designed and optimized a CRISPR interference-mediated pooled library of essential genes (CIMPLE) approaching coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to an uncharacterized bacterial growth inhibitor structurally different from antibiotics and discovered that it inhibits the essential peptidyl-tRNA hydrolase. Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action.},
}

*Carboxylic Ester Hydrolases/metabolism/genetics
Burkholderia cenocepacia/genetics/drug effects
Anti-Bacterial Agents/pharmacology
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Library
Bacterial Proteins/metabolism/genetics
Enzyme Inhibitors/pharmacology
Genes, Essential
Mutation

@article {pmid39527477,
year = {2024},
author = {Wang, J and Du, J and Luo, X and Guo, L and Liu, Y and Zhou, J and Zou, Y and Lu, Z and Pan, X and Chen, X and Zhong, A and Wan, X and Wang, L and Liu, H and Dai, S and Zhang, S and Xiong, X and Tan, P and Wang, M and Wu, B and Zhang, Q and Wang, Y and Zhang, M and Lu, R and Lin, H and Li, Y and Li, Y and Han, Z and Chen, L and Hu, B and Liu, Y and Na, F and Chen, C},
title = {A platform of functional studies of ESCC-associated gene mutations identifies the roles of TGFBR2 in ESCC progression and metastasis.},
journal = {Cell reports},
volume = {43},
number = {11},
pages = {114952},
doi = {10.1016/j.celrep.2024.114952},
pmid = {39527477},
issn = {2211-1247},
mesh = {Animals ; *Receptor, Transforming Growth Factor-beta Type II/genetics/metabolism ; Humans ; Mice ; *Mutation/genetics ; *Disease Progression ; *Esophageal Squamous Cell Carcinoma/genetics/pathology/metabolism ; *Smad3 Protein/metabolism/genetics ; *Neoplasm Metastasis ; Esophageal Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {Genomics studies have detected numerous genetic alterations in esophageal squamous cell carcinoma (ESCC). However, the functions of these mutations largely remain elusive, partially due to a lack of feasible animal models. Here, we report a convenient platform with CRISPR-Cas9-mediated introduction of genetic alterations and orthotopic transplantation to generate a series of primary ESCC models in mice. With this platform, we validate multiple frequently mutated genes, including EP300, FAT1/2/4, KMT2D, NOTCH2, and TGFBR2, as tumor-suppressor genes in ESCC. Among them, TGFBR2 loss dramatically promotes tumorigenesis and multi-organ metastasis. Paradoxically, TGFBR2 deficiency leads to Smad3 activation, and disruption of Smad3 partially restrains the progression of Tgfbr2-mutated tumors. Drug screening with tumor organoids identifies that pinaverium bromide represses Smad3 activity and restrains Tgfbr2-deficient ESCC. Our studies provide a highly efficient platform to investigate the in vivo functions of ESCC-associated mutations and develop potential treatments for this miserable malignancy.},
}

Animals
*Receptor, Transforming Growth Factor-beta Type II/genetics/metabolism
Humans
Mice
*Mutation/genetics
*Disease Progression
*Esophageal Squamous Cell Carcinoma/genetics/pathology/metabolism
*Smad3 Protein/metabolism/genetics
*Neoplasm Metastasis
Esophageal Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
CRISPR-Cas Systems/genetics

@article {pmid39509270,
year = {2024},
author = {Jin, M and Peng, Y and Peng, J and Yu, S and Wu, C and Yang, X and Zhu, J and Infante, O and Xu, Q and Wang, H and Wu, K and Xiao, Y},
title = {A supergene controls facultative diapause in the crop pest Helicoverpa armigera.},
journal = {Cell reports},
volume = {43},
number = {11},
pages = {114939},
doi = {10.1016/j.celrep.2024.114939},
pmid = {39509270},
issn = {2211-1247},
mesh = {Animals ; *Moths/genetics/physiology ; Insect Proteins/genetics/metabolism ; Chromosome Inversion ; Diapause, Insect/genetics ; CRISPR-Cas Systems/genetics ; Diapause/genetics ; Crops, Agricultural/genetics/parasitology ; Helicoverpa armigera ; },
abstract = {Many insect species, including the economically important pest Helicoverpa armigera, avoid unfavorable conditions by suspending development. This form of phenotypic plasticity-facultative diapause-is a complex trait, though its evolution and intricate genetic architecture remain poorly understood. To investigate how such a polygenic trait could be locally adapted, we explore its genetic architecture. We map a large-effect diapause-associated locus to the Z chromosome by crossing high- and low-latitude populations. By generating multiple chromosome-scale assemblies, we identify an ∼5.93-Mb chromosomal inversion that constitutes the locus. Within this inversion, 33 genes harbor divergent non-synonymous mutations, notably including three circadian rhythm genes: Period, Clock, and Cycle. CRISPR-Cas9 knockout experiments confirm that each gene is independently essential for pupal diapause. Thus, a diapause supergene arose within H. armigera via a Z chromosome inversion, enabling local climatic adaptation in this economically important crop pest.},
}

Animals
*Moths/genetics/physiology
Insect Proteins/genetics/metabolism
Chromosome Inversion
Diapause, Insect/genetics
CRISPR-Cas Systems/genetics
Diapause/genetics
Crops, Agricultural/genetics/parasitology
Helicoverpa armigera

@article {pmid39607301,
year = {2024},
author = {Du, B and Chen, M and Chang, L and Zhang, X and Zhang, X and Wang, X and Gong, P and Zhang, N and Zhang, X and Li, X and Li, J},
title = {Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the Neospora caninum challenge.},
journal = {Virulence},
volume = {15},
number = {1},
pages = {2427844},
doi = {10.1080/21505594.2024.2427844},
pmid = {39607301},
issn = {2150-5608},
mesh = {Animals ; *Neospora/genetics/immunology/pathogenicity ; *Coccidiosis/prevention & control/immunology/parasitology/veterinary ; *Mice, Inbred C57BL ; Mice ; *Protozoan Proteins/genetics/immunology ; *Protozoan Vaccines/immunology/genetics ; *Vaccines, Attenuated/immunology/genetics/administration & dosage ; *Gene Knockout Techniques ; Female ; Immunization ; Virulence ; CRISPR-Cas Systems ; },
abstract = {Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.},
}

Animals
*Neospora/genetics/immunology/pathogenicity
*Coccidiosis/prevention & control/immunology/parasitology/veterinary
*Mice, Inbred C57BL
Mice
*Protozoan Proteins/genetics/immunology
*Protozoan Vaccines/immunology/genetics
*Vaccines, Attenuated/immunology/genetics/administration & dosage
*Gene Knockout Techniques
Female
Immunization
Virulence
CRISPR-Cas Systems

@article {pmid39606944,
year = {2024},
author = {Gautier, LJ},
title = {CRISPR in 3D: Innovations in Disease Modelling and Personalized Medicine.},
journal = {BioTechniques},
volume = {},
number = {},
pages = {1-6},
doi = {10.1080/07366205.2024.2418748},
pmid = {39606944},
issn = {1940-9818},
abstract = {The CRISPR-Cas system of genetic engineering has had a significant impact on science and society since its advent in 2013. CRISPR integration with 3D culture systems such as organ-on-a-chips, as well as fast emerging commercial technologies, has encouraged translation of more complex pathophysiological modelling and personalized medicine.[Formula: see text].},
}

@article {pmid39606390,
year = {2024},
author = {Orosco, C and Rananaware, SR and Huang, B and Hanna, MP and Ahmadimashhadi, MR and Lewis, JG and Baugh, MP and Bodin, AP and Flannery, SJ and Lange, IH and Fang, ZR and Karalkar, VN and Meister, KS and Jain, PK},
title = {DNA-guided CRISPR/Cas12 for RNA targeting.},
journal = {medRxiv : the preprint server for health sciences},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.11.21.24317744},
pmid = {39606390},
abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.},
}

@article {pmid39605323,
year = {2024},
author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Cardenas, CLL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Lawton, J and Kim, N and Silverstein, RA and Ferreira da Silva, J and de la Cruz, D and Richa, R and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL},
title = {In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.11.11.621817},
pmid = {39605323},
issn = {2692-8205},
abstract = {Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.},
}

@article {pmid39605316,
year = {2024},
author = {Weber, R and Vasella, F and Klimko, A and Silginer, M and Lamfers, M and Neidert, MC and Regli, L and Schwank, G and Weller, M},
title = {Targeting the IDH1 [R132H] mutation in gliomas by CRISPR/Cas precision base editing.},
journal = {Neuro-oncology advances},
volume = {6},
number = {1},
pages = {vdae182},
pmid = {39605316},
issn = {2632-2498},
abstract = {BACKGROUND: Gliomas, the most frequent malignant primary brain tumors, lack curative treatments. Understanding glioma-specific molecular alterations is crucial to develop novel therapies. Among them, the biological consequences of the isocitrate dehydrogenase 1 gene mutation (IDH1 [R132H]) remain inconclusive despite its early occurrence and widespread expression.

METHODS: We thus employed CRISPR/Cas adenine base editors, which allow precise base pair alterations with minimal undesirable effects, to correct the IDH1 [R132H] mutation.

RESULTS: Successful correction of the IDH1 [R132H] mutation in primary patient-derived cell models led to reduced IDH1 [R132H] protein levels and decreased production of 2-hydroxyglutarate, but increased proliferation. A dual adeno-associated virus split intein system was used to successfully deliver the base editor in vitro and in vivo.

CONCLUSIONS: Taken together, our study provides a strategy for a precise genetic intervention to target the IDH1 [R132H] mutation, enabling the development of accurate models to study its impact on glioma biology and serving as a framework for an in vivo gene therapy.},
}

@article {pmid39604746,
year = {2024},
author = {Saha, UB and Dixit, KK and Jadhav, SV and Pathak, KN and Gupta, NS and Saroj, SD},
title = {Genomic Insights of Multidrug-Resistant Enterococcus faecalis and Acinetobacter baumannii Isolated from a Sepsis Patient with Pauci-Immune Crescentic Glomerulonephritis, India.},
journal = {Current microbiology},
volume = {82},
number = {1},
pages = {16},
pmid = {39604746},
issn = {1432-0991},
support = {SR/PURSE/2023/181//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {Humans ; *Acinetobacter baumannii/genetics/drug effects/isolation & purification ; *Drug Resistance, Multiple, Bacterial/genetics ; *Enterococcus faecalis/genetics/drug effects/isolation & purification ; *Genome, Bacterial ; *Anti-Bacterial Agents/pharmacology ; India ; *Sepsis/microbiology ; *Acinetobacter Infections/microbiology ; *Glomerulonephritis/microbiology/genetics ; Gram-Positive Bacterial Infections/microbiology ; Whole Genome Sequencing ; Microbial Sensitivity Tests ; Genomics ; Coinfection/microbiology ; Bacteremia/microbiology ; },
abstract = {Acinetobacter baumannii and Enterococcus faecalis are opportunistic bacteria frequently associated with hospital-acquired infections. A. baumannii nosocomial infections in intensive care units are a worldwide problem, with high mortality rates. It may also develop rapidly multidrug resistance (MDR), extensive drug resistance (XDR), and even pan-drug resistance (PDR). Colistin resistance which is an example of pan-drug resistance, is highly alarming as it's used as a last-line antibiotic. Microbes capable of crossing epithelial barriers such as E. faecalis have developed novel strategies to counter antimicrobial agents and cause bacteremia in immunocompromised patients. However, the coinfection of these bacteria in the same patient is unusual. Here, we report a genomic investigation of the extensively drug-resistant E. faecalis and A. baumannii isolated from the blood sample of a patient diagnosed with pauci-immune crescentic glomerulonephritis (PICGN). Identification of cultures isolated from blood sample was carried out using whole-genome sequencing and resistome profiles were mapped. Whole genome sequencing revealed that E. faecalis SVJ-EF01 had a genome size of 2,935,226 bp and GC content of 37.4%, whereas A. baumannii SVJ-AC01 had a genome size of 3,730,857 bp and GC content of 39%. Draft genomes were functionally annotated demonstrating that the organism harbors multiple virulence factors and antimicrobial-resistant mechanisms including MDR efflux pumps. A. baumannii genome possessed a CRISPR-Cas system which might contribute to antimicrobial resistance. This highlights the significance of polymicrobial nature in ESKAPE pathogenesis research. This genomic investigation helps to gain insights into the virulence, resistance profile, and functional potential of these pathogens.},
}

Humans
*Acinetobacter baumannii/genetics/drug effects/isolation & purification
*Drug Resistance, Multiple, Bacterial/genetics
*Enterococcus faecalis/genetics/drug effects/isolation & purification
*Genome, Bacterial
*Anti-Bacterial Agents/pharmacology
India
*Sepsis/microbiology
*Acinetobacter Infections/microbiology
*Glomerulonephritis/microbiology/genetics
Gram-Positive Bacterial Infections/microbiology
Whole Genome Sequencing
Microbial Sensitivity Tests
Genomics
Coinfection/microbiology
Bacteremia/microbiology

@article {pmid39515197,
year = {2024},
author = {Tabibian, M and Ghafouri-Fard, S},
title = {CRISPR-mediated silencing of non-coding RNAs: A novel putative treatment for prostate cancer.},
journal = {Pathology, research and practice},
volume = {264},
number = {},
pages = {155710},
doi = {10.1016/j.prp.2024.155710},
pmid = {39515197},
issn = {1618-0631},
mesh = {Humans ; *Prostatic Neoplasms/genetics/pathology/therapy ; Male ; *CRISPR-Cas Systems/genetics ; RNA, Untranslated/genetics ; Gene Silencing ; Gene Expression Regulation, Neoplastic/genetics ; },
abstract = {Non-coding RNAs affect carcinogenic processes in diverse tissues, such as prostate. Several of these transcripts act as oncogenes driving prostate cancer. Thus, they are putative targets for treatment of this type of cancer. CRISPR/Cas9 technology has provided new tools for modulation of expression of these oncogenes in order to combat several aspects of carcinogenesis, including invasion cascades and metastasis. This review aimed to describe novel achievements in modulation of expression of non-coding RNAs using CRISPR/Cas9 technology in prostate cancer.},
}

Humans
*Prostatic Neoplasms/genetics/pathology/therapy
Male
*CRISPR-Cas Systems/genetics
RNA, Untranslated/genetics
Gene Silencing
Gene Expression Regulation, Neoplastic/genetics

@article {pmid38808710,
year = {2025},
author = {Easwaran, M and Madasamy, S and Venkidasamy, B},
title = {Theranostic Potential of Bacteriophages against Oral Squamous Cell Carcinoma.},
journal = {Current gene therapy},
volume = {25},
number = {2},
pages = {89-91},
pmid = {38808710},
issn = {1875-5631},
mesh = {Humans ; *Mouth Neoplasms/therapy ; *Bacteriophages/genetics ; *Carcinoma, Squamous Cell/therapy ; *Theranostic Nanomedicine/methods ; Phage Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Oral Squamous Cell Carcinoma (OSCC) is a widespread and challenging disease that accounts for 94% of cancers of the oral cavity worldwide. Bacteriophages (phages) have shown promise as a potential theranostic agent for the treatment of OSCC. It may offer advantages in overcoming the challenges of conventional methods. Modern high-throughput pyrosequencing techniques confirm the presence of specific bacterial strains associated with OSCC. Bio-panning and filamentous phages facilitate visualization of the peptide on surfaces and show high affinity in OSCC cells. The peptide has the potential to bind integrin (αvβ6), aid in diagnosis, and inhibit the proliferation of OSCC cells. Mimotopes of tumor-associated antigens show cytotoxic and immune responses against cancer cells. Biomarker-based approaches such as transferrin enable early OSCC diagnosis. A modified temperate phage introduces CRISPR-Cas3 to target antimicrobial-resistant bacteria associated with OSCC. The research findings highlight the evolving field of phage diagnostics and therapy and represent a new avenue for non-invasive, targeted approaches to the detection and treatment of OSCC. However, extensive clinical research is required to validate the efficacy of phages in innovative cancer theranostic strategies.},
}

Humans
*Mouth Neoplasms/therapy
*Bacteriophages/genetics
*Carcinoma, Squamous Cell/therapy
*Theranostic Nanomedicine/methods
Phage Therapy/methods
CRISPR-Cas Systems

@article {pmid39604418,
year = {2024},
author = {Qiao, L and Niu, L and Wang, M and Wang, Z and Kong, D and Yu, G and Ye, H},
title = {A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {10310},
pmid = {39604418},
issn = {2041-1723},
support = {no.32430064//National Natural Science Foundation of China (National Science Foundation of China)/ ; no.32261160373//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Genetic Therapy/methods ; *Optogenetics/methods ; Mice ; *Dependovirus/genetics ; Humans ; *Disease Models, Animal ; Metabolic Diseases/therapy/genetics ; Light ; Insulin/metabolism ; Cytokines/metabolism ; Obesity/therapy/genetics/metabolism ; Diabetes Mellitus, Type 1/therapy/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Male ; Mice, Inbred C57BL ; Blood Glucose/metabolism ; Gene Expression Regulation ; Tumor Necrosis Factor Ligand Superfamily Member 14 ; },
abstract = {Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We use the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.},
}

Animals
*Genetic Therapy/methods
*Optogenetics/methods
Mice
*Dependovirus/genetics
Humans
*Disease Models, Animal
Metabolic Diseases/therapy/genetics
Light
Insulin/metabolism
Cytokines/metabolism
Obesity/therapy/genetics/metabolism
Diabetes Mellitus, Type 1/therapy/genetics/metabolism
CRISPR-Cas Systems
HEK293 Cells
Male
Mice, Inbred C57BL
Blood Glucose/metabolism
Gene Expression Regulation
Tumor Necrosis Factor Ligand Superfamily Member 14

@article {pmid39604381,
year = {2024},
author = {Goldberg, GW and Kogenaru, M and Keegan, S and Haase, MAB and Kagermazova, L and Arias, MA and Onyebeke, K and Adams, S and Beyer, DK and Fenyö, D and Noyes, MB and Boeke, JD},
title = {Engineered transcription-associated Cas9 targeting in eukaryotic cells.},
journal = {Nature communications},
volume = {15},
number = {1},
pages = {10287},
pmid = {39604381},
issn = {2041-1723},
support = {F32GM137482//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; K99GM147604//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM118851//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; RM1HG009491//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Transcription, Genetic ; *Saccharomyces cerevisiae/genetics/metabolism ; Gene Editing/methods ; DNA/metabolism/genetics ; Eukaryotic Cells/metabolism ; HEK293 Cells ; Genetic Engineering/methods ; },
abstract = {DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.},
}

Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
*Transcription, Genetic
*Saccharomyces cerevisiae/genetics/metabolism
Gene Editing/methods
DNA/metabolism/genetics
Eukaryotic Cells/metabolism
HEK293 Cells
Genetic Engineering/methods

@article {pmid39604306,
year = {2024},
author = {Kim, Y and Lee, E and Kang, BC},
title = {Etiolation promotes protoplast transfection and genome editing efficiency.},
journal = {Physiologia plantarum},
volume = {176},
number = {6},
pages = {e14637},
doi = {10.1111/ppl.14637},
pmid = {39604306},
issn = {1399-3054},
support = {RS-2022-NR072144//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Lactuca/genetics/growth & development ; *Transfection/methods ; Brassica/genetics/growth & development ; Genome, Plant/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism/genetics ; },
abstract = {In plants, DNA-free genome editing using preassembled clustered regularly interspaced short palindromic repeats (CRISPR)-ribonucleoprotein (RNP) has the advantage of avoiding transgene integration and limiting off-target effects. The efficiency of this gene editing strategy can vary, so optimization of protoplast transfection conditions is necessary to achieve maximum yield. In this study, we examined the effects of etiolation, or increased exposure to darkness during cultivation, on the transfection efficiency of protoplasts from lettuce and Chinese cabbage. Seedlings were grown under three different conditions: non-etiolated, etiolated, and de-etiolated. First, we tested PEG-mediated transfection after etiolation using a plasmid DNA for green fluorescent protein (GFP)-expression. Etiolated protoplasts had the highest percentage of GFP-expressing cells, with a 3.1-fold and 4.8-fold improvement in lettuce and Chinese cabbage, respectively, compared with non-etiolated protoplasts. We also assessed gene editing of endogenous genes after etiolation using CRISPR-RNP. Using targeted deep sequencing, we observed the highest editing efficiency in etiolated protoplasts from both plant species, for the LsPDS and LsFT genes in lettuce, this led to an 8.7-fold and 4.4-fold improvement compared with non-etiolated protoplasts, respectively. These results suggest that etiolation during seedling growth can improve transfection efficiency and DNA-free gene editing in protoplasts.},
}

*Protoplasts/metabolism
*Gene Editing/methods
*Lactuca/genetics/growth & development
*Transfection/methods
Brassica/genetics/growth & development
Genome, Plant/genetics
CRISPR-Cas Systems
Green Fluorescent Proteins/metabolism/genetics

@article {pmid39603754,
year = {2024},
author = {Maladan, Y and Retnaningrum, E and Daryono, BS and Sarassari, R and Sari, RF and Balqis, SA and Wahid, GA and Safari, D},
title = {A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9-Targeted Sequencing.},
journal = {The Journal of molecular diagnostics : JMD},
volume = {26},
number = {12},
pages = {1045-1054},
doi = {10.1016/j.jmoldx.2024.08.009},
pmid = {39603754},
issn = {1943-7811},
mesh = {*Streptococcus pneumoniae/genetics/classification ; *CRISPR-Cas Systems/genetics ; *Serotyping/methods ; Humans ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; Pneumococcal Infections/microbiology/genetics ; Genome, Bacterial ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.},
}

*Streptococcus pneumoniae/genetics/classification
*CRISPR-Cas Systems/genetics
*Serotyping/methods
Humans
High-Throughput Nucleotide Sequencing/methods
Sequence Analysis, DNA/methods
Pneumococcal Infections/microbiology/genetics
Genome, Bacterial

@article {pmid39601843,
year = {2024},
author = {Cao, L and Chen, W and Kang, W and Lei, C and Nie, Z},
title = {Engineering stimuli-responsive CRISPR-Cas systems for versatile biosensing.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {39601843},
issn = {1618-2650},
support = {22034002//National Natural Science Foundation of China/ ; 92253304//National Natural Science Foundation of China/ ; 2020YFA0907500//National Key Research and Development Program of China/ ; 2021YFA0910100//National Key Research and Development Program of China/ ; },
abstract = {The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.},
}

@article {pmid39601181,
year = {2024},
author = {Bamundo, M and Palumbo, S and D'Auria, L and Missero, C and Di Girolamo, D},
title = {CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology-Directed Repair Mutagenesis.},
journal = {Current protocols},
volume = {4},
number = {11},
pages = {e70056},
pmid = {39601181},
issn = {2691-1299},
mesh = {Humans ; *Keratinocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Ribonucleoproteins/genetics/metabolism ; Transfection/methods ; Recombinational DNA Repair ; Cells, Cultured ; Mutagenesis ; },
abstract = {Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex Basic Protocol 2: Deletion of specific genomic region using RNPs via nucleofection Basic Protocol 3: Use of homology-directed repair system to introduce site-specific mutations.},
}

Humans
*Keratinocytes/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Gene Knockout Techniques/methods
Ribonucleoproteins/genetics/metabolism
Transfection/methods
Recombinational DNA Repair
Cells, Cultured
Mutagenesis

@article {pmid39599550,
year = {2024},
author = {Mikhaylova, Y and Tyumentseva, M and Karbyshev, K and Tyumentsev, A and Slavokhotova, A and Smirnova, S and Akinin, A and Shelenkov, A and Akimkin, V},
title = {Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.},
journal = {Pathogens (Basel, Switzerland)},
volume = {13},
number = {11},
pages = {},
pmid = {39599550},
issn = {2076-0817},
support = {075-15-2019-1666//Ministry of Science and Higher Education of Russian Federation/ ; },
abstract = {Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.},
}

@article {pmid39597141,
year = {2024},
author = {Xie, S and Yue, Y and Yang, F},
title = {Recent Advances in CRISPR/Cas System-Based Biosensors for the Detection of Foodborne Pathogenic Microorganisms.},
journal = {Micromachines},
volume = {15},
number = {11},
pages = {},
pmid = {39597141},
issn = {2072-666X},
support = {32102633//National Natural Science Foundation of China/ ; },
abstract = {Foodborne pathogens pose significant risks to food safety. Conventional biochemical detection techniques are facing a series of challenges. In recent years, with the gradual development of CRISPR (clustered regularly interspaced short palindromic repeats) technology, CRISPR/Cas system-based biosensors, a newly emerging technology, have received much attention from researchers because of their supreme flexibility, sensitivity, and specificity. While numerous CRISPR-based biosensors have a broad application in the field of environmental monitoring, food safety, and point-of-care diagnosis, they remain in high demand to summarize recent advances in CRISPR/Cas system-based biosensors for foodborne pathogen detection. In this paper, we briefly classify and discuss the working principles of CRISPR/Cas systems with trans-cleavage activity in applications for the detection of foodborne pathogenic microorganisms. We highlight the current status, the unique feature of each CRISPR system and CRISPR-based biosensing platforms, and the integration of CRISPR-Cas with other techniques, concluding with a discussion of the advantages, disadvantages, and future directions.},
}

@article {pmid39596692,
year = {2024},
author = {Chen, J and Miao, Z and Kong, D and Zhang, A and Wang, F and Liu, G and Yu, X and Luo, L and Liu, Y},
title = {Application of CRISPR/Cas9 Technology in Rice Germplasm Innovation and Genetic Improvement.},
journal = {Genes},
volume = {15},
number = {11},
pages = {},
pmid = {39596692},
issn = {2073-4425},
support = {23N11900100//Shanghai Municipal Commission of Science and technology/ ; INV-033236-3//The Bill and Melinda Gates Foundation/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Crops, Agricultural/genetics ; Genome, Plant ; },
abstract = {Improving the efficiency of germplasm innovation has always been the aim of rice breeders. Traditional hybrid breeding methods for variety selection rarely meet the practical needs of rice production. The emergence of genome-editing technologies, such as CRISPR/Cas9, provides a new approach to the genetic improvement of crops such as rice. The number of published scientific papers related to "gene editing" and "CRISPR/Cas9" retrievable on websites both from China and other countries exhibited an increasing trend, year by year, from 2014 to 2023. Research related to gene editing in rice accounts for 33.4% and 12.3% of all the literature on gene editing published in China and other countries, respectively, much higher than that on maize and wheat. This article reviews recent research on CRISPR/Cas9 gene-editing technology in rice, especially germplasm innovation and genetic improvement of commercially promoted varieties with improved traits such as disease, insect, and herbicide resistance, salt tolerance, quality, nutrition, and safety. The aim is to provide a reference for the precise and efficient development of new rice cultivars that meet market demand.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Breeding/methods
Plants, Genetically Modified/genetics
Crops, Agricultural/genetics
Genome, Plant

@article {pmid39578577,
year = {2024},
author = {Ingle, H and Molleston, JM and Hall, PD and Bui, D and Wang, L and Bhatt, KD and Foster, L and Antia, A and Ding, S and Lee, S and Fremont, DH and Baldridge, MT},
title = {The neonatal Fc receptor is a cellular receptor for human astrovirus.},
journal = {Nature microbiology},
volume = {9},
number = {12},
pages = {3321-3331},
pmid = {39578577},
issn = {2058-5276},
support = {R01AI127552//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI139314//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI181955//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI141478//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32AI106688//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32DK077653//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; F30AI181285//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI150796//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; U19AI116484//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R00 AI141683/AI/NIAID NIH HHS/United States ; Pathogenesis of Infectious Disease Program//Burroughs Wellcome Fund (BWF)/ ; Pew Biomedical Scholars Program//Pew Charitable Trusts/ ; T32DK007130//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; P20 GM109035/GM/NIGMS NIH HHS/United States ; 75N93022C00035/AI/NIAID NIH HHS/United States ; R00 AI141683/AI/NIAID NIH HHS/United States ; P20 GM109035/GM/NIGMS NIH HHS/United States ; 75N93022C00035/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Fc/metabolism/genetics ; *Histocompatibility Antigens Class I/metabolism/genetics ; *Mamastrovirus/metabolism/genetics/physiology ; *Virus Internalization ; *Dipeptidyl Peptidase 4/metabolism/genetics ; Astroviridae Infections/virology/metabolism ; CRISPR-Cas Systems ; Cell Line ; Receptors, Virus/metabolism ; HEK293 Cells ; },
abstract = {Human astroviruses (HAstV) are major causes of gastroenteritis, especially in children, and there are no vaccines or antivirals currently available. Little is known about host factors required for their cellular entry. Here we utilized complementary CRISPR-Cas9-based knockout and activation screens to identify neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection in vitro. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding of FcRn, but not DPP4, with HAstV virions and the purified spike protein. This suggests that FcRn is a receptor for HAstVs while DPP4 is a cofactor for entry. Inhibitors for DPP4 and FcRn currently in clinical use prevented HAstV infection in cell lines and human enteroids. Our results reveal mechanisms of HAstV entry as well as druggable targets to limit HAstV infection.},
}

Humans
*Receptors, Fc/metabolism/genetics
*Histocompatibility Antigens Class I/metabolism/genetics
*Mamastrovirus/metabolism/genetics/physiology
*Virus Internalization
*Dipeptidyl Peptidase 4/metabolism/genetics
Astroviridae Infections/virology/metabolism
CRISPR-Cas Systems
Cell Line
Receptors, Virus/metabolism
HEK293 Cells

@article {pmid39482441,
year = {2024},
author = {Lo, YL and Hong, CJ and Wang, CS and Yang, CP},
title = {Modulating versatile pathways using a cleavable PEG shell and EGFR-targeted nanoparticles to deliver CRISPR-Cas9 and docetaxel for triple-negative breast cancer inhibition.},
journal = {Archives of pharmacal research},
volume = {47},
number = {10-11},
pages = {829-853},
pmid = {39482441},
issn = {1976-3786},
support = {NSTC 113-2320-B-A49-032//National Science and Technology Council/ ; MOST 110-2320-B-A49A-510-MY3//National Science and Technology Council/ ; MOST 107-2320-B-010-015-MY3//National Science and Technology Council/ ; },
mesh = {*Triple Negative Breast Neoplasms/drug therapy/pathology/metabolism/genetics ; Animals ; *Docetaxel/pharmacology/administration & dosage ; Humans ; *ErbB Receptors/antagonists & inhibitors/genetics/metabolism ; *Polyethylene Glycols/chemistry ; *Antineoplastic Agents/pharmacology/chemistry/administration & dosage ; Mice ; Female ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry ; Cell Line, Tumor ; Mice, Nude ; Mice, Inbred BALB C ; ELAV-Like Protein 1/metabolism/genetics/antagonists & inhibitors ; Drug Delivery Systems ; Cell Proliferation/drug effects ; },
abstract = {Human antigen R (HuR), an RNA-binding protein, is implicated in regulating mRNA stability and translation in cancer, especially in triple-negative breast cancer (TNBC), a highly aggressive form. CRISPR/Cas9-mediated HuR knockout (HuR CRISPR) presents a promising genetic therapeutic approach, but it encounters transfection limitations. Docetaxel (DTX), an effective cytotoxic agent against metastatic breast cancer (BC), faces challenges related to vehicle-associated adverse events in DTX formulations. Therefore, we designed multifunctional nanoparticles with pH-sensitive PEG derivatives and targeting peptides to enable efficient HuR CRISPR and DTX delivery to human TNBC MDA-MB-231 cells and tumor-bearing mice. Our findings indicated that these nanoparticles displayed pH-responsive cytotoxicity, precise EGFR targeting, efficient tumor penetration, successful endosomal escape, and accurate nuclear and cytoplasmic localization. They also demonstrated the ability to spare normal cells and prevent hemolysis. Our study concurrently modulated multiple pathways, including EGFR, Wnt/β-catenin, MDR, and EMT, through the regulation of EGFR/PI3K/AKT, HuR/galectin-3/GSK-3β/β-catenin, and P-gp/MRPs/BCRP, as well as YAP1/TGF-β/ZEB1/Slug/MMPs. The combined treatment arrested the cell cycle at the G2 phase and inhibited EMT, effectively impeding tumor progression. Tissue distribution, biochemical assays, and histological staining revealed the enhanced safety profile of pH-responsive PEG- and peptide-modified nanoformulations in TNBC mice. The DTX-embedded and peptide-modified nanoparticles mitigated the side effects of DTX, enhanced cytotoxicity in TNBC MDA-MB-231 cells, and exhibited remarkable antitumor efficacy and safety in TNBC-bearing mice with HuR CRISPR deletion. Collectively, the combination therapy of DTX and CRISPR/Cas9 offers an effective platform for delivering antineoplastic agents and gene-editing systems to combat tumor resistance and progression in TNBC.},
}

*Triple Negative Breast Neoplasms/drug therapy/pathology/metabolism/genetics
Animals
*Docetaxel/pharmacology/administration & dosage
Humans
*ErbB Receptors/antagonists & inhibitors/genetics/metabolism
*Polyethylene Glycols/chemistry
*Antineoplastic Agents/pharmacology/chemistry/administration & dosage
Mice
Female
*CRISPR-Cas Systems
*Nanoparticles/chemistry
Cell Line, Tumor
Mice, Nude
Mice, Inbred BALB C
ELAV-Like Protein 1/metabolism/genetics/antagonists & inhibitors
Drug Delivery Systems
Cell Proliferation/drug effects

@article {pmid39431866,
year = {2024},
author = {Qiao, Y and Wang, X and Kang, X and Song, Y and Zhang, J and Han, Q},
title = {A chemiluminescent sensor based on CRISPR-HCR technology for the hypersensitive detection of Mycobacterium tuberculosis.},
journal = {Analytical methods : advancing methods and applications},
volume = {16},
number = {46},
pages = {7927-7939},
doi = {10.1039/d4ay01517g},
pmid = {39431866},
issn = {1759-9679},
mesh = {*Mycobacterium tuberculosis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA, Bacterial/analysis ; *Luminescent Measurements/methods ; Tuberculosis/diagnosis ; Bacterial Proteins/genetics ; Biosensing Techniques/methods ; Limit of Detection ; DNA, Single-Stranded/genetics/chemistry ; CRISPR-Associated Proteins/genetics ; Sputum/microbiology ; Endodeoxyribonucleases/genetics ; },
abstract = {Tuberculosis is a highly infectious bacterial disease caused by Mycobacterium tuberculosis. The spread of this agent has caused serious health problems worldwide, and the rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the trans cleavage ability of CRISPR-Cas12a against single-stranded DNA was combined with hybridization chain reaction and chemiluminescent signal to establish an imaging sensor for the hypersensitive detection of M. tuberculosis DNA. We observed linear relationships between the concentration of M. tuberculosis DNA and the output signal over the ranges of 10 to 200 pM and 200 to 800 pM DNA. The equations of the standard curves were y = 56.08x + 3303, with R[2] = 0.9916 for the lower range and y = 15.69x + 10 685, with R[2] = 0.9929 for the higher range. The limit of detection was as low as 0.83 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 1 copy per μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from sputum of hospitalized tuberculosis patients. The sensitivity and specificity of the proposed sensor is combined with a long shelf-life and a low cost of materials. This study introduces a new method for tuberculosis detection and broadens the application of CRISPR-Cas12a-based sensors in clinical diagnosis.},
}

*Mycobacterium tuberculosis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
*DNA, Bacterial/analysis
*Luminescent Measurements/methods
Tuberculosis/diagnosis
Bacterial Proteins/genetics
Biosensing Techniques/methods
Limit of Detection
DNA, Single-Stranded/genetics/chemistry
CRISPR-Associated Proteins/genetics
Sputum/microbiology
Endodeoxyribonucleases/genetics

@article {pmid39271536,
year = {2024},
author = {Ko, EJ and Suh, DS and Kim, H and Lee, JY and Eo, WK and Kim, H and Kim, KH and Cha, HJ},
title = {Transcriptome analysis of the effect of HERV-K env gene knockout in ovarian cancer cell lines.},
journal = {Genes & genomics},
volume = {46},
number = {11},
pages = {1293-1301},
pmid = {39271536},
issn = {2092-9293},
support = {Biomedical Research Institute Grant (20230143)//Pusan National University Hospital/ ; },
mesh = {Humans ; *Ovarian Neoplasms/genetics/virology ; Female ; *Endogenous Retroviruses/genetics ; Cell Line, Tumor ; *Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; Transcriptome/genetics ; Gene Expression Profiling/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Human endogenous retroviruses (HERVs) have been implicated in the pathogenesis of various diseases, particularly cancers. Previous investigations from our group demonstrated that targeted knockout (KO) of the HERV-K env gene led to a significant reduction in tumorigenic attributes, including proliferation, migration, and invasion of ovarian cancer cells.

OBJECTIVE: In this study, we aimed to elucidate the impact of HERV-K env KO on gene expression in ovarian cancer cell lines through comparative RNA sequencing (RNA-Seq) analysis with two distinct HERV-K env KO ovarian cancer cell lines, SKOV3 and OVCAR3.

METHODS: HERV-K env gene KO was achieved in SKOV3 and OVCAR3 ovarian cancer cell lines using the CRISPR-Cas9 system. Next-generation mRNA sequencing was employed to assess the gene expression profiles of both mock and HERV-K env KO ovarian cancer cells. Furthermore, comprehensive analyses involving gene ontology and pathway assessments were conducted.

RESULTS: Transcriptome analysis revealed that 23 differentially expressed genes (DEGs) were upregulated and 17 DEGs were downregulated in SKOV3 cells. In OVCAR3 cells, 198 DEGs were upregulated, and 17 DEGs were downregulated. Notably, 53 DEGs exhibited statistically significant differences among the 1,612 DEGs identified. Our findings indicate that HERV-K env gene KO exerts a profound influence on gene expression patterns in OVCAR3 cells, while genetic alterations in expression were relatively modest in SKOV3 cells. Nevertheless, genes ND1, ND2, and CYTB displayed a common increase in expression, while ERRFI1 and NDRG1 exhibited a decrease in expression in both cell lines.

CONCLUSION: Our study demonstrates that KO of the HERV-K env gene in ovarian cancer cell lines has a substantial impact on gene expression patterns and can be used to identify potential therapeutic targets for ovarian cancer and related diseases.},
}

Humans
*Ovarian Neoplasms/genetics/virology
Female
*Endogenous Retroviruses/genetics
Cell Line, Tumor
*Gene Knockout Techniques
Gene Expression Regulation, Neoplastic
Transcriptome/genetics
Gene Expression Profiling/methods
CRISPR-Cas Systems

@article {pmid39596534,
year = {2024},
author = {Kim, JY and Lee, YJ and Lee, HJ and Go, JY and Lee, HM and Park, JS and Cho, YG and Jung, YJ and Kang, KK},
title = {Knockout of OsGAPDHC7 Gene Encoding Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Affects Energy Metabolism in Rice Seeds.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212470},
pmid = {39596534},
issn = {1422-0067},
mesh = {*Oryza/genetics/metabolism/growth & development/enzymology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism ; *Energy Metabolism/genetics ; *Gene Expression Regulation, Plant ; *Seeds/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Cytosol/metabolism ; CRISPR-Cas Systems ; Starch/metabolism ; Carbohydrate Metabolism/genetics ; },
abstract = {Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified in the rice genome. Among them, the expression level of five genes of cytosolic GAPDH was shown to be different for each organ. The mutation induction of the GAPDHC7 gene by the CRISPR/Cas9 system revealed that the 7 bp and 2 bp deletion, early end codon, was used in protein production. In addition, the selected mutants showed lower plant heights compared to the wild-type plants. To investigate the effect on carbohydrate metabolism, the expression of the genes of starch-branched enzyme I (SbeI), sucrose synthase (SS), and 3-phosphoglycer phosphokinase (PGK) increased the expression of the SBeI gene threefold in the knockout lines compared to the wild-type (WT) plant, while the expression of the SS and PGK genes decreased significantly. And the starch and soluble sugar content of the knockout lines increased by more than 60% compared to the WT plant. Also, the free amino acid content was significantly increased in the Gln and Asn contents of the knockout lines compared to the WT plants, while the contents of Gly and Ser were decreased. Our results suggest that OsGAPDHC7 has a great influence on energy metabolism, such as pre-harvested sprouting and amino acid content.},
}

*Oryza/genetics/metabolism/growth & development/enzymology
*Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism
*Energy Metabolism/genetics
*Gene Expression Regulation, Plant
*Seeds/metabolism/genetics
Plant Proteins/genetics/metabolism
Gene Knockout Techniques
Cytosol/metabolism
CRISPR-Cas Systems
Starch/metabolism
Carbohydrate Metabolism/genetics

@article {pmid39596521,
year = {2024},
author = {Sheveleva, O and Protasova, E and Grigor'eva, E and Butorina, N and Kuziaeva, V and Antonov, D and Melnikova, V and Medvedev, S and Lyadova, I},
title = {The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212456},
pmid = {39596521},
issn = {1422-0067},
support = {№075-15-2021-1075//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Interferon-beta/metabolism/genetics ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Embryoid Bodies/metabolism/cytology ; Cell Line ; Gene Editing/methods ; },
abstract = {Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells, here, we aimed to generate human iPSCs overexpressing interferon-beta (IFNB, IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-β. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation, IFNB-iPSCs formed embryoid bodies and upregulated endoderm, mesoderm, and some ectoderm markers. However, an upregulation of key neuroectoderm markers, PAX6 and LHX2, was compromised. A negative effect of IFN-β on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-β. The study describes new IFN-β-producing iPSC lines suitable for the generation of various types of IFN-β-producing cells for future experimental and clinical applications, and it unravels an inhibitory effect of IFN-β on stem cell neuroectoderm differentiation.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Interferon-beta/metabolism/genetics
*Cell Differentiation/genetics
*CRISPR-Cas Systems
Genetic Engineering/methods
Embryoid Bodies/metabolism/cytology
Cell Line
Gene Editing/methods

@article {pmid39596457,
year = {2024},
author = {Sakovina, L and Vokhtantsev, I and Akhmetova, E and Vorobyeva, M and Vorobjev, P and Zharkov, DO and Novopashina, D},
title = {Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212392},
pmid = {39596457},
issn = {1422-0067},
support = {22-14-00294//Russian Science Foundation/ ; 121031300042-1//Russian state-funded project/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; },
abstract = {The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min[-1] and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
CRISPR-Associated Protein 9/metabolism/genetics
Humans
Ultraviolet Rays
Photolysis
Light

@article {pmid39596448,
year = {2024},
author = {Chang, H and Sha, H and Gao, S and Liu, Q and Liu, Y and Ma, C and Shi, B and Nie, S},
title = {A Novel Gene, OsRLCK191, Involved in Culm Strength Improving Lodging Resistance in Rice.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212382},
pmid = {39596448},
issn = {1422-0067},
support = {LH2021C092, CZKYF2023-1-A005, CXSTOP2021005, HTRC202209//Heilongjiang Provincial Natural Science Foundation of China, Fundamental Research Funds for the Research Institutes of Heilongjiang Province, the Opening Foundation of the Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Co/ ; },
mesh = {*Oryza/genetics/growth & development ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Cell Wall/metabolism/genetics ; CRISPR-Cas Systems ; Genes, Plant ; Mutation ; },
abstract = {Lodging is one of the major problems in rice production. However, few genes that can explain the culm strength within the temperate japonica subspecies have been identified. In this study, we identified OsRLCK191, which encodes receptor-like cytoplasmic kinase and plays critical roles in culm strength. OsRLCK191 mutants were produced by the CRISPR-Cas9 DNA-editing system. Compared with wild types (WTs), the bending moment of the whole plant (WP), the bending moment at breaking (BM), and the section modulus (SM) were decreased in rlck191 significantly. Although there is no significant decrease in the culm length of rlck191 compared with the WT; in the mutant, except the length of the fourth internode being significantly increased, the lengths of other internodes are significantly shortened. In addition, the yield traits of panicle length, thousand-seed weight, and seed setting rate decreased significantly in rlck191. Moreover, RNA-seq experiments were performed at an early stage of rice panicle differentiation in shoot apex. The differentially expressed genes (DEGs) are mainly involved in cell wall biogenesis, cell wall polysaccharide metabolic processes, cellar component biogenesis, and DNA-binding transcription factors. Transcriptome analysis of the cell wall biological process pathways showed that major genes that participated in the cytokinin oxidase/dehydrogenase family, cellulose synthase catalytic subunit genes, and ethylene response factor family transcription factor were related to culm strength. Our research provides an important theoretical basis for analyzing the lodging resistance mechanism and lodging resistance breeding of temperate japonica.},
}

*Oryza/genetics/growth & development
*Plant Proteins/genetics/metabolism
*Gene Expression Regulation, Plant
Cell Wall/metabolism/genetics
CRISPR-Cas Systems
Genes, Plant
Mutation

@article {pmid39596447,
year = {2024},
author = {Meng, L and Zhang, J and Clarke, N},
title = {A Critical Review of Recent Advances in Maize Stress Molecular Biology.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212383},
pmid = {39596447},
issn = {1422-0067},
support = {C200937//Natural Science Foundation of Heilongjiang Province/ ; },
mesh = {*Zea mays/genetics/metabolism ; *Stress, Physiological/genetics ; *Gene Expression Regulation, Plant ; Genome-Wide Association Study ; Quantitative Trait Loci ; Droughts ; },
abstract = {With the intensification of global climate change and environmental stress, research on abiotic and biotic stress resistance in maize is particularly important. High temperatures and drought, low temperatures, heavy metals, salinization, and diseases are widespread stress factors that can reduce maize yields and are a focus of maize-breeding research. Molecular biology provides new opportunities for the study of maize and other plants. This article reviews the physiological and biochemical responses of maize to high temperatures and drought, low temperatures, heavy metals, salinization, and diseases, as well as the molecular mechanisms associated with them. Special attention is given to key transcription factors in signal transduction pathways and their roles in regulating maize stress adaptability. In addition, the application of transcriptomics, genome-wide association studies (GWAS), and QTL technology provides new strategies for the identification of molecular markers and genes for maize-stress-resistance traits. Crop genetic improvements through gene editing technologies such as the CRISPR/Cas system provide a new avenue for the development of new stress-resistant varieties. These studies not only help to understand the molecular basis of maize stress responses but also provide important scientific evidence for improving crop tolerance through molecular biological methods.},
}

*Zea mays/genetics/metabolism
*Stress, Physiological/genetics
*Gene Expression Regulation, Plant
Genome-Wide Association Study
Quantitative Trait Loci
Droughts

@article {pmid39596426,
year = {2024},
author = {Niu, D and Zhao, Q and Xu, L and Lin, K},
title = {Physiological and Molecular Mechanisms of Lepidopteran Insects: Genomic Insights and Applications of Genome Editing for Future Research.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212360},
pmid = {39596426},
issn = {1422-0067},
support = {2022YFD1400600//Research on Mechanisms of Grassland Moth Catastrophes and Sustainable Control Technologies/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Lepidoptera/genetics ; *Genomics/methods ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Genome, Insect ; Insecticides/pharmacology ; },
abstract = {Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.},
}

Animals
*Gene Editing/methods
*Lepidoptera/genetics
*Genomics/methods
*Insecticide Resistance/genetics
CRISPR-Cas Systems
Genome, Insect
Insecticides/pharmacology

@article {pmid39596412,
year = {2024},
author = {Sosnovtseva, AO and Demidova, NA and Klimova, RR and Kovalev, MA and Kushch, AA and Starodubova, ES and Latanova, AA and Karpov, DS},
title = {Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212346},
pmid = {39596412},
issn = {1422-0067},
support = {22-14-00377//Russian Science Foundation/ ; 075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Herpesvirus 1, Human/genetics ; *Herpes Simplex/diagnosis/therapy/virology ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Gene Editing/methods ; Drug Resistance, Viral/genetics ; Genetic Therapy/methods ; },
abstract = {It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.},
}

*CRISPR-Cas Systems
Humans
*Herpesvirus 1, Human/genetics
*Herpes Simplex/diagnosis/therapy/virology
Animals
Antiviral Agents/therapeutic use/pharmacology
Gene Editing/methods
Drug Resistance, Viral/genetics
Genetic Therapy/methods

@article {pmid39596282,
year = {2024},
author = {Khmeleva, SA and Kurbatov, LK and Ptitsyn, KG and Timoshenko, OS and Morozova, DD and Suprun, EV and Radko, SP and Lisitsa, AV},
title = {Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR/Cas13a Analysis of NASBA Amplicons.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212218},
pmid = {39596282},
issn = {1422-0067},
support = {075-15-2021-1345, unique identifier RF-193021X0012//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Solanum tuberosum/microbiology ; *CRISPR-Cas Systems ; *Clavibacter/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology ; RNA, Ribosomal, 16S/genetics ; Plant Tubers/microbiology ; Molecular Diagnostic Techniques ; },
abstract = {The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)-the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the "one-pot" format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.},
}

*Solanum tuberosum/microbiology
*CRISPR-Cas Systems
*Clavibacter/genetics
*Nucleic Acid Amplification Techniques/methods
*Plant Diseases/microbiology
RNA, Ribosomal, 16S/genetics
Plant Tubers/microbiology
Molecular Diagnostic Techniques

@article {pmid39596246,
year = {2024},
author = {Shmelev, ME and Pilnik, AA and Shved, NA and Penkova, AO and Gulaia, VS and Kumeiko, VV},
title = {IDH1 R132H and TP53 R248Q Mutations Modulate Glioma Cell Migration and Adhesion on Different ECM Components.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212178},
pmid = {39596246},
issn = {1422-0067},
support = {20-15-00378п//Russian Science Foundation/ ; },
mesh = {Humans ; *Isocitrate Dehydrogenase/genetics ; *Cell Movement/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; *Extracellular Matrix/metabolism/genetics ; Cell Line, Tumor ; *Cell Adhesion/genetics ; *Glioma/genetics/pathology/metabolism ; Mutation ; Brain Neoplasms/genetics/pathology/metabolism ; },
abstract = {Mutations in IDH1 and TP53 have a significant impact on glioma prognosis and progression; however, their roles in tumor cell invasion in terms of interactions with particular components of the extracellular matrix (ECM) are still unclear. Using gene editing protocol based on CRISPR-Cas 9 with cytidine deaminase, we introduced point mutations into U87MG glioblastoma cells to establish modified cell lines with heterozygous IDH1 R132H, homozygous TP53 R248Q and heterozygous IDH1 R132H, homozygous TP53 R248Q genotypes. A comparative study of cell migration on major ECM components was carried out by high-content microscopy. IDH1 R132H mutation introduced to U87MG glioblastoma cells was shown to decrease the migration speed on Matrigel and collagen IV substrates compared to the wild-type. This data were supported by cell adhesion quantification via the lateral shift assay performed by atomic force microscopy (AFM). TP53 R248Q mutation increased cell adhesion to various substrates and significantly promoted cell migration on hyaluronic acid and chondroitin sulfate but did not change the migration rates on laminin and collagens IV and I. A double-mutant genotype produced by consequently introducing IDH1 R132H and TP53 R248Q to parental glioblastoma cells was characterized by the highest migration among all the cell lines, with particularly faster motility on chondroitin sulfate. These findings underscore the complex interactions between glioma cells, with the most important driver mutations and specific ECM components regulating cancer cell migration, offering valuable insights for potential therapeutic targets in glioma treatment.},
}

Humans
*Isocitrate Dehydrogenase/genetics
*Cell Movement/genetics
*Tumor Suppressor Protein p53/genetics/metabolism
*Extracellular Matrix/metabolism/genetics
Cell Line, Tumor
*Cell Adhesion/genetics
*Glioma/genetics/pathology/metabolism
Mutation
Brain Neoplasms/genetics/pathology/metabolism

@article {pmid39596176,
year = {2024},
author = {Wang, Z and Wang, C and Zhai, Y and Bai, Y and Wang, H and Rong, X},
title = {Loss of Brcc3 in Zebrafish Embryos Increases Their Susceptibility to DNA Damage Stress.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212108},
pmid = {39596176},
issn = {1422-0067},
support = {2022YFC2804400//the National Key R & D Program of China/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Zebrafish Proteins/genetics/metabolism ; *DNA Damage ; Tumor Suppressor Protein p53/genetics/metabolism ; Ultraviolet Rays/adverse effects ; Apoptosis/genetics ; CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; DNA Breaks, Double-Stranded ; DNA Repair ; Etoposide/pharmacology ; },
abstract = {DNA double-strand breaks (DSBs) represent one of the most severe forms of genetic damage in organisms, yet vertebrate models capable of monitoring DSBs in real-time remain scarce. BRCA1/BRCA2-containing complex subunit 3 (BRCC3), also known as BRCC36, functions within various multiprotein complexes to mediate diverse biological processes. However, the physiological role of BRCC3 in vertebrates, as well as the underlying mechanisms that govern its activity, are not well understood. To explore these questions, we generated brcc3-knockout zebrafish using CRISPR/Cas9 gene-editing technology. While brcc3 mutant zebrafish appear phenotypically normal and remain fertile, they exhibit significantly increased rates of mortality and deformity following exposure to DNA damage. Furthermore, embryos lacking Brcc3 display heightened p53 signaling, elevated γ-H2AX levels, and increased apoptosis in response to DNA-damaging agents such as ultraviolet (UV) light and Etoposide (ETO). Notably, genetic inactivation of p53 or pharmacological inhibition of Ataxia-telangiectasia mutated (ATM) activity rescues the hypersensitivity to UV and ETO observed in Brcc3-deficient embryos. These findings suggest that Brcc3 plays a critical role in DNA damage response (DDR), promoting cell survival during embryogenesis. Additionally, brcc3-null mutant zebrafish offer a promising vertebrate model for real-time monitoring of DSBs.},
}

Animals
*Zebrafish/genetics/embryology
*Zebrafish Proteins/genetics/metabolism
*DNA Damage
Tumor Suppressor Protein p53/genetics/metabolism
Ultraviolet Rays/adverse effects
Apoptosis/genetics
CRISPR-Cas Systems
Embryo, Nonmammalian/metabolism
DNA Breaks, Double-Stranded
DNA Repair
Etoposide/pharmacology

@article {pmid39596160,
year = {2024},
author = {de Jonge, AV and Csikós, T and Eken, M and Bulthuis, EP and Poddighe, PJ and Roemer, MGM and Chamuleau, MED and Mutis, T},
title = {Delineating MYC-Mediated Escape Mechanisms from Conventional and T Cell-Redirecting Therapeutic Antibodies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212094},
pmid = {39596160},
issn = {1422-0067},
mesh = {Humans ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism/immunology ; Killer Cells, Natural/immunology/metabolism ; Antibodies, Bispecific/pharmacology ; Apoptosis ; Rituximab/pharmacology/therapeutic use ; Multiple Myeloma/therapy/immunology/genetics/pathology ; CRISPR-Cas Systems ; Tumor Escape/drug effects ; Antigens, CD20/immunology/metabolism/genetics ; Antibodies, Monoclonal/pharmacology/therapeutic use ; },
abstract = {In B-cell malignancies, the overexpression of MYC is associated with poor prognosis, but its mechanism underlying resistance to immunochemotherapy remains less clear. In further investigations of this issue, we show here that the pharmacological inhibition of MYC in various lymphoma and multiple myeloma cell lines, as well as patient-derived primary tumor cells, enhances their susceptibility to NK cell-mediated cytotoxicity induced by conventional antibodies targeting CD20 (rituximab) and CD38 (daratumumab), as well as T cell-mediated cytotoxicity induced by the CD19-targeting bispecific T-cell engager blinatumomab. This was associated with upregulation of the target antigen only for rituximab, suggesting additional escape mechanisms. To investigate these mechanisms, we targeted the MYC gene in OCI-LY18 cells using CRISPR-Cas9 gene-editing technology. CRISPR-Cas9-mediated MYC targeting not only upregulated CD20 but also triggered broader apoptotic pathways, upregulating pro-apoptotic PUMA and downregulating anti-apoptotic proteins BCL-2, XIAP, survivin and MCL-1, thereby rendering tumor cells more prone to apoptosis, a key tumor-lysis mechanism employed by T-cells and NK-cells. Moreover, MYC downregulation boosted T-cell activation and cytokine release in response to blinatumomab, revealing a MYC-mediated T-cell suppression mechanism. In conclusion, MYC overexpressing tumor cells mitigated the efficacy of therapeutic antibodies through several non-overlapping mechanisms. Given the challenges associated with direct MYC inhibition due to toxicity, successful modulation of MYC-mediated immune evasion mechanisms may improve the outcome of immunotherapeutic approaches in B-cell malignancies.},
}

Humans
*T-Lymphocytes/immunology/metabolism
Cell Line, Tumor
Proto-Oncogene Proteins c-myc/genetics/metabolism/immunology
Killer Cells, Natural/immunology/metabolism
Antibodies, Bispecific/pharmacology
Apoptosis
Rituximab/pharmacology/therapeutic use
Multiple Myeloma/therapy/immunology/genetics/pathology
CRISPR-Cas Systems
Tumor Escape/drug effects
Antigens, CD20/immunology/metabolism/genetics
Antibodies, Monoclonal/pharmacology/therapeutic use

@article {pmid39596103,
year = {2024},
author = {Begovic, M and Schneider, L and Zhou, X and Hamdani, N and Akin, I and El-Battrawy, I},
title = {The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252212034},
pmid = {39596103},
issn = {1422-0067},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Channelopathies/genetics/pathology ; *Myocytes, Cardiac/metabolism/pathology ; Brugada Syndrome/genetics/pathology/metabolism ; Gene Editing ; Animals ; Tachycardia, Ventricular/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype-phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Channelopathies/genetics/pathology
*Myocytes, Cardiac/metabolism/pathology
Brugada Syndrome/genetics/pathology/metabolism
Gene Editing
Animals
Tachycardia, Ventricular/genetics/pathology
CRISPR-Cas Systems

@article {pmid39595965,
year = {2024},
author = {Fernández, JP and Petersen, B and Hassel, P and Lucas Hahn, A and Kielau, P and Geibel, J and Kues, WA},
title = {Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs.},
journal = {International journal of molecular sciences},
volume = {25},
number = {22},
pages = {},
doi = {10.3390/ijms252211894},
pmid = {39595965},
issn = {1422-0067},
support = {TRR 127//Deutsche Forschungsgemeinschaft/ ; },
mesh = {Animals ; *Electroporation/methods ; *Microinjections/methods ; Swine ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Antigens, Heterophile/genetics/immunology ; Galactosyltransferases/genetics ; Embryo, Mammalian/metabolism ; Mosaicism ; Blastocyst/metabolism ; Zygote/metabolism ; Mixed Function Oxygenases ; },
abstract = {In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.},
}

Animals
*Electroporation/methods
*Microinjections/methods
Swine
*CRISPR-Cas Systems
*Gene Editing/methods
*Gene Knockout Techniques/methods
Animals, Genetically Modified
Antigens, Heterophile/genetics/immunology
Galactosyltransferases/genetics
Embryo, Mammalian/metabolism
Mosaicism
Blastocyst/metabolism
Zygote/metabolism
Mixed Function Oxygenases