期刊
CELL REPORTS
卷 30, 期 9, 页码 2859-+出版社
CELL PRESS
DOI: 10.1016/j.celrep.2020.02.010
关键词
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类别
资金
- Sanjana laboratory
- NYU startup fund
- NYGC startup fund
- NIH/National Human Genome Research Institute (NHGRI) [R00HG008171, DP2HG010099]
- NIH/National Cancer Institute (NCI) [R01CA218668]
- Defense Advanced Research Projects Agency (DARPA) [D18AP00053]
- Sidney Kimmel Foundation
- Brain and Behavior Foundation
- Hope Funds for Cancer Research postdoctoral fellowship
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.
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