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Cas9 - Springer Nature

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Last Updated: 10 September 2022

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A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer

Castration-resistant prostate cancer is characterized by gene mutations that are frequent in castration-resistant prostate cancer, allowing for polymerase inhibitor therapy. However, therapy resistance is a significant medical issue, and genes that contribute to PARPi resistance are poorly understood. Low ARH3 genotyping of 126 prostate cancer patients revealed low ARH3 expression as an independent predictor of recurrence. Our results contribute to the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC, as well as a new model of PARPi resistance by reduced autophagy.

Source link: https://doi.org/10.1038/s41388-022-02427-2


Temperature dependent in vitro binding and release of target DNA by Cas9 enzyme

When loaded with the guide RNA sequence, CRISPRu2013Cas9 cleaves the DNA at the target location as indicated by the guide RNA sequence. We investigate the binding properties of Cas9 enzyme to a sequence of DNA at a variety of temperatures and, surprisingly, find that the Cas9 enzyme can identify and bind its target DNA at temperatures as low as 4 u00b0C. The cleaved DNA products remain strongly bound to the Cas9 enzyme, although the enzyme is released from the enzyme only at higher temperatures, according to a study published in the journal. We find the rate of Cas9 binding to target DNA to target DNA to be 0. 8 0. 2 minu22121 at 37 u00b0C using the gel shift assays. We also tested denaturant dependent release of a cleaved product, which revealed a similar manufacturing pattern with a dissociation constant of 0. 23 mM.

Source link: https://doi.org/10.1038/s41598-022-19485-x


Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks

DNA tests can be determined by error-free or mutagenic repair, according to single cell reporters who can determine whether DNA breaks are fixed by error-free or mutagenic repair. Multiple pathways are used to restore error-free repair, mutation, and genomic damage, with results ranging from error-free repair to genetic mutation, and genomic loss. To address this, we designed and tested three fluorescent Cas9-based reporters, DSB-Spectrum, that simultaneously measure multiple DNA repair pathways at a DSB. DSB-repair by error-free canonical non-homologous end-joining, mutationagenic repair versus HR, and mutagenic end-joining versus single strand annealing versus HR is distinguishable by DSB-Spectrum reporters. Our results show that SSA-mediated DSB-repair also occurs at endogenous genomic loci, owing to Alu elements or homologous gene regions. When both pathways compete for the same substrate, we find that long-range end-resection factors DNA2 and Exo1 contribute to SSA and HR. These Cas9-based DSB-Spectrum reporters aid in the detailed review of repair pathway crosstalk and the DSB-repair findings. These new Cas9-based DSB-Spectrum reporters support the thorough review of repair pathway crosstalk and DSB-repair findings.

Source link: https://doi.org/10.1038/s41467-022-32743-w


High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs

Despite the availability of Cas9 variants with various protospacer-adjacent motif compatibilities, some genomic lociu2014, particularly those with pyrimidine-rich PAM sequences, remain inaccessible by high-activity Cas9 proteins. Variants eNme2-T. 1 and eNme2-T. 2 are available as existing variants of N_4TN PAM sequences with comparable editing results, while eNme2-C and eNme2-C. NR's eNme2-T. 1 and eNme2-T. 2 have less stringent PAM specifications, comparable or higher engagement in a variety of human cell types, as well as lower off-target application at N_4CN PAM sequences. New compact Cas9 variants that aim for the majority single pyrimidine nucleotide PAMs are produced by directed evolution.

Source link: https://doi.org/10.1038/s41587-022-01410-2


Fusing an exonuclease with Cas9 enhances homologous recombination in Pichia pastoris

Results The endogenous exonuclease Mre11 and Exo1 had the highest success rates in seamless deletion of FAA1 in continuous deletion, and transferring the MRE11 to the C-terminal of CAS9 had the highest positive rate and a large number of clones. When overexpressing RAD52, we found that CAS9-MRE11 expression significantly increased positive rates. Conclusions in P : Mre11's endogenous exonuclease Mre11 with Cas9 improves homologous recombination rate.

Source link: https://doi.org/10.1186/s12934-022-01908-z


Massively parallel genomic perturbations with multi-target CRISPR interrogates Cas9 activity and DNA repair at endogenous sites

Generic Cas9 binding and cleavage results were published by a rapid post-cleavage Cas9 fragmentation and repair factor gene expression at protospacer adjacent motif-proximal genomic DNA, revealing immediate post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Additionally, mgRNAs reported that Cas9 binding is increased at chromatin-accessible locations, and cleavage by bound Cas9 is more effective near transcribed regions, with confounding effects from guide RNA sequence. mgRNAs also enabled high-throughput analysis of Cas9 response to double-strand breaks with high temporal fidelity, revealing the presence, extent, and kinetics of reversible DNA damage-induced chromatin decompaction. This work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our knowledge of intracellular Cas9 activity and DNA damage response at endogenous loci, as well as mouse genetic fingerprints.

Source link: https://doi.org/10.1038/s41556-022-00975-z


Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.)

Backgrounds The cucumber's very limited genetic basis makes breeding of this species difficult. The CRISPR/Cas9 system is characterized by its simplicity, low cost, and high quality, which has opened a new path for cucumber functional genetics and cucumber mocular breeding. The explants were slightly scratched after cutting, pre-cultured for one day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could possibly have a significant rise in the GUS staining rate of explants. Three and two PCR positive lines were obtained from 210 and 207 explants respectively, and the corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were developed and transformed using the above-optimized cucumber genetic transformation device. In the Cas9-sgRNA-1 transformed three PCR positive lines, no sequence mutation at the target loci of CsGCN5 was discovered. Both PCR positive lines indicated that a mutant line with targeted homozygous mutation was discovered from the Cas9-sgRNA-2 transformed two PCR positive lines.

Source link: https://doi.org/10.1007/s11033-022-07558-z


CRISPR-Cas9 based stress tolerance: New hope for abiotic stress tolerance in chickpea (Cicer arietinum)

Traditional breeding has been used in the past to produce crop plants conferring resistance to stresses, but newer approaches are ineffective in producing greater improvement. In addition, the introduction of chickpea transgenic lines after knocking out of 4CL and REV7 genes shows drought tolerance, which provides the foundation for future studies in chickpea. This review article explores gene function based on the CRISPR-Cas9 for the production of abiotic stress-tolerant crop plants, emphasizing the chickpea to spark the new efforts of breeding abiotic stress-tolerant chickpea varieties.

Source link: https://doi.org/10.1007/s11033-022-07391-4


Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome

CRISPR/Cas9 has emerged as a simple yet powerful gene editing device that can be used to produce targeted mutations in crops plant genes. Agrobacterium tumefaciens, a simple and cost-delivery device in plant cells, has been used for the creation of CRISPR/Cas9-mediated mutations in crop plants, including potato. In the current research, the green fluorescent protein transgene was first detected in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with integrated gfp copy integrated and another with four gfp copies integrated, were exposed to CRISPR/Cas9-mediated mutations in the transgene using three different gRNAs. During the entire culture/regeneration process, the GFP fluorescence disappearance was tracked. Although all three gRNAs successfully knocked out the transgene, their performance varied notably and did not entirely match those predicted by some guide RNA prediction software.

Source link: https://doi.org/10.1007/s11240-022-02310-8


Evaluation of CRISPR/Cas9 System Effects on Knocking Out NEAT1 Gene in AGS Gastric Cancer Cell Line with Therapeutic Perspective

Aim Gastric cancer is one of the most common malignant tumors worldwide, with an increasing incidence rate. In various cells, nuclear speckle assembly transcript 1 is a long non-coding RNA that controls cell cycle progression, apoptosis, cell proliferation, and migration. The present survey was carried out to determine the effects of NEAT1 gene knocking out by the CRISPR/Cas9 procedure in human gastric cancer cells. Methods The CRISPR/Cas9 genome editing method was used to knock out NEAT1 in AGS cells as a gastric cancer model. Knockout of NEAT1 by implication on miR-34a gene expression resulted in AGS cell apoptosis and total apoptosis of increasing FAS level and total apoptosis. Conclusions: NEAT1 plays a crucial role in the cellular processes of GCu2019s appearance and may be a new therapeutic target in GC, according to the findings.

Source link: https://doi.org/10.1007/s12029-021-00669-z

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions