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

Autor: Sangree AK; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Griffith AL; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Szegletes ZM; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Roy P; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., DeWeirdt PC; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Hegde M; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., McGee AV; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Hanna RE; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA., Doench JG; Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames St., Cambridge, MA, USA. jdoench@broadinstitute.org.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2022 Mar 14; Vol. 13 (1), pp. 1318. Date of Electronic Publication: 2022 Mar 14.
DOI: 10.1038/s41467-022-28884-7
Abstrakt: 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
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