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• W3010221330 startingPage "2859" @default.
• W3010221330 abstract "A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants." @default.
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• W3010221330 date "2020-03-01" @default.
• W3010221330 modified "2023-10-14" @default.
• W3010221330 title "High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation" @default.
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• W3010221330 doi "https://doi.org/10.1016/j.celrep.2020.02.010" @default.
• W3010221330 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7558435" @default.
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