A highly specific SpCas9 variant is identified by in vivo screening in yeast

Nature Biotechnology 36, 265 (2018). doi:10.1038/nbt.4066 Authors: Antonio Casini, Michele Olivieri, Gianluca Petris, Claudia Montagna, Giordano Reginato, Giulia Maule, Francesca Lorenzin, Davide Prandi, Alessandro Romanel, Francesca Demichelis, Alberto Inga & Anna Cereseto Despite the utility of CRISPR–Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. 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 (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 ad...
Source: Nature Biotechnology - Category: Biotechnology Authors: Tags: Research Source Type: research