Cas9
基因组编辑
清脆的
生物
计算生物学
引导RNA
遗传学
基因
作者
Antonio Casini,Michele Olivieri,Gianluca Petris,Claudia Montagna,Giordano Reginato,Giulia Maule,Francesca Lorenzin,Davide Prandi,Alessandro Romanel,Francesca Demichelis,Alberto Inga,Anna Cereseto
摘要
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 purposes1,2. 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 variants3,4 (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.
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