Additionally, to expand the targeting-scope of SpCas9, several groups have created PAM-flexible variants. Several high-fidelity versions of SpCas9, including SpCas9-HF1 8, HypaCas9 9, and eSpCas9-1.1 10, developed by rational design, and HiFi Cas9 11 and evoCas9 12, identified by randomized screening in bacteria and yeast, have been shown to attenuate off-target cutting without substantial loss of on-target activity. However, off-target activity may occur due to recognition of an unanticipated protospacer adjacent motif (PAM) or tolerance of mispairing between the guide and DNA 5, 6, 7. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.Ĭoupling CRISPR technology to highly parallel methods to write and read DNA 1, 2, 3, as well as viral technologies that enable delivery to nearly any cell type of interest, has enabled pooled genetic screens across diverse models, assays, and fields 4. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology.
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