In:
Nature Communications, Springer Science and Business Media LLC, Vol. 13, No. 1 ( 2022-03-14)
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.
Type of Medium:
Online Resource
ISSN:
2041-1723
DOI:
10.1038/s41467-022-28884-7
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2022
detail.hit.zdb_id:
2553671-0
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