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Cas9 - Crossref

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Last Updated: 10 September 2022

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

Abstract The CRISPR-associated protein 9 system has proven to be a versatile tool for genome editing in a variety of in vivo and in vitro applications. We investigated the binding properties of Cas9 enzyme to the sequence specific target DNA at a variety of temperatures and discovered and bound its target DNA at 90 % b0C in this study, but in our analysis, we discovered that the Cas9 enzyme can identify and bind its target DNA at temperatures as low as 90 °C. 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 this journal. We calculate the rate of Cas9 binding to target DNA at 37 % at 37 0. 05 0. 05 min u22121 at 37 °C using the gel shift assays. 01.

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


Implications of CRISPR-Cas9 in developing Next Generation Biofuel: A Minireview

The key drawbacks of biofuel production at the commercial level are their low yield, non-availability of feedstock, feedback inhibition, the presence of inhibitory pathways in many animals, and biofuel intolerance of organisms. Gene knockout and gene cassette inserts employing CRISPR-Cas9 in Saccharomyces cerevisiae and Kluyveromyces marxianus have resulted in increased bioethanol and 2-Phenyl ethanol production in these species, respectively. Several bacterial strains' genomes were also modified to improve ethanol and butanol production in them. The biosynthetic pathways of lignin in plant-based biofuel production hindered the safe release of fermentable sugars, thereby impeded effective biofuel production.

Source link: https://doi.org/10.2174/1389203723666220907110310


Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing

We used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions in nine DM2 patients in the single-nucleotide level. Interestingly, only two DM2 samples displayed the expected pure CCTG repeat pattern, while the other seven displayed TCTG blocks at the 3'u2032 end, which had not been reported in DM2 patients before, but not in DM2 patients.

Source link: https://doi.org/10.7554/elife.80229


Identifying Context-specific Network Features for CRISPR-Cas9 Targeting Efficiency Using Accurate and Interpretable Deep Neural Network

Machine learning has been used to improve sgRNA selection, but several challenges remain. The success of predictive models is limited by the amount of available data in many cell lines, confusion of gene network function and its variable effects on phenotype, and elusive biological interpretation of computational models. In addition to the last three nucleotides in sgRNA 3u2019end, we also discovered that gene context-specific network properties are important for prediction accuracy. Our results will help develop more accurate predictive models of CRISPR-Cas9 in a variety of biological disorders as well as effective and safe gene therapy.

Source link: https://doi.org/10.1101/505602


Targeted dual base editing with Campylobacter jejuni Cas9 by a single AAV-mediated delivery

Here, we have produced cjCas9-based base editors, including a cytosine base editor and an adenine base editor that can successfully induce endogenous base substitutions in HEK293T cells by up to 91. 2% at the HPD gene. The active windows of 13 endogenous target sites, according to a review of the base editing effectiveness of 13 endogenous target websites revealed that cjCBEmax and cjABE8e's active windows were larger than those of spCas9-based base editors', and that their specificities were marginally smaller than that of cjCas9. Engineered cjCas9 and gRNA scaffold can raise the base editing yield of cjABE8e by up to 6. 4 percent in HEK293T cells.

Source link: https://doi.org/10.21203/rs.3.rs-1973343/v1


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

Abstract u2013b Abstract Abstract: Despite the availability of Cas9 versions with varying protospacer-adjacent motif compatibilities, some genomic loci u2014, particularly those with pyrimidine-rich PAM sequences, remain inaccessible by high-activity Cas9 proteins. In addition, increasing PAM sequence compatibility via engineering can increase off-target activity. We used this information to produce high-activity variants of eNme2-T. 1, eNme2-T. 2, eNme2-T. 2, eNme2-C, and eNme2-C. NR. We used this information to produce the eNme2-T. 1, eNme2-T. 2, eNme2-C. NR. Variants eNme2-T. 1 and eNme2-T. 2 provide access to N 4 TN PAM sequences with comparable editing results as existing versions, while eNme2-C and eNme2-C. NR's eNme2-T. 1 and eNme2-T. 1 incorporate less stringent PAM specifications, comparable or higher deployment in a variety of human cell types, as well as lower off-target operation at N 4 CN PAM sequences.

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


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

Abstract DNA double-strand breaks are repaired by a multitude of paths, with results ranging from error-free repair to mutation and genomic loss. DSB-repair by error-free canonical non-homologous end-joining versus homologous recombination, mutationagenic repair versus HR, and mutagenic end-joining versus single strand annealing versus HR - distinguishing between DSB-repair and HR versus HR reveals the difference between DSB-repair by error-free canonical end-joining versus HR versus HR otrum versus homologistic end-logistic end-homologious end-joining versus homologous re recombination versus HR versus HR versus HR versus HR versus HR versus HR annealing versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus HR versus Our results show that SSA-mediated DSB-repair also occurs at endogenous genomic loci, characterized by Alu elements or homologous gene regions. Since both pathways compete for the same substrate, we find that long-range end-resection factors DNA2 and Exo1 have both contributed to SSA and lower HR.

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


Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice

We developed a strategy for targeted mutagenesis and golden SNP replacement of rice's LcyE gene in rice using the CRSPR/Cas9 and the geminiviral replicon. We have used geminiviral replicon amplification to obtain a substantial amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand defect in the target gene by homology-directed repair. Our aim was the LcyE allele derived from anther culture containing a golden SNP replacement. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6. 8 percent higher than that of the wild-type calli, respectively. In the LcyEsg2-HDR1 and LcyEsg2-HDR2 series, the reactive oxygen species content was lower. These results show that effective HDR can be achieved in the golden SNP replacement using a single and modular system adaptable to various rice goals and other crops.

Source link: https://doi.org/10.3390/ijms231810383

* 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