Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2

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Abstract

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. 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. 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. 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. 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.

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Sangree, A. K., Griffith, A. L., Szegletes, Z. M., Roy, P., DeWeirdt, P. C., Hegde, M., … Doench, J. G. (2022). Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-28884-7

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