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A highly specific SpCas9 variant is identified by in vivo screening in yeast
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A highly specific SpCas9 variant is identified by in vivo screening in yeast
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Journal Article
A highly specific SpCas9 variant is identified by in vivo screening in yeast
2018
Overview
Evolved SpCas9 variant evoCas9 has improved specificity and retains near wild-type on-target activity.
Despite the utility of CRISPR–Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes
1
,
2
. We developed a yeast-based assay to identify optimized
Streptococcus pyogenes
Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants
3
,
4
(fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ 13/109
/ 13/31
/ 38/22
/ 38/23
/ 38/70
/ 42/44
/ 45/77
/ Biomedical Engineering/Biotechnology
/ Cleavage
/ CRISPR
/ CRISPR-Associated Protein 9 - genetics
/ CRISPR-Cas Systems - genetics
/ Editing
/ Genomes
/ Kinases
/ letter
/ Methods
/ Mutation
/ Nuclease
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ Streptococcus pyogenes - enzymology
/ Streptococcus pyogenes - genetics
/ Yeast
