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.
Highlights
Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology
For each Cas9 variant we systematically investigated on-target activity: cutting efficiency at intended sequences; and off-target activity: cleavage at unintended sites
After calculating LFCs relative to plasmid DNA (pDNA), we found that replicates were well-correlated (Pearson’s r = 0.77–0.99 C > T base editors (CBEs) screens, Supplementary Data 5; 0.77–0.95 A > G base editors (ABEs), Supplementary Data 6), and we averaged the data across the cell lines
Summary
Numerous rationally-designed and directed-evolution variants of SpCas have been reported to expand the utility of CRISPR technology. SpG was developed to recognize NG PAMs, while SpRY has been characterized as essentially PAM-less18 Together, these variants enable targeting of genomic loci previously inaccessible by wildtype (WT) SpCas. Legut et al. and Kim et al. assayed xCas9-3.7 and Cas9-NG alongside WT-Cas in pooled screens The former used a flow cytometry-based assay targeting three cell-surface genes with guides using all possible 3 nucleotide PAMs, the latter used a library-on-library approach to profile PAM preference. Both found that Cas9-NG is more active at NGH sites than WT-Cas, but that PAM flexibility comes at the cost of reduced efficacy. We uncover LOF mutations in BRCA1, and demonstrate the utility of this approach for mapping drug-target interactions by resistance screens with Venetoclax and BCL2
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