Mitigation of off-target effects in CRISPR genome editing

The CRISPR/Cas9 system demonstrates unparalleled editing efficiency, however, cleavage of genomic DNA at sites with an imperfect match to the guide RNA (gRNA) can occur as an undesired off-target effect (OTE). The magnitude of these OTEs is highest using expression-based systems like plasmids or viruses. Use of chemically-modified synthetic guide RNAs delivered into cells pre-bound to recombinant Cas9 protein as a ribonucleoprotein (RNP) complex offers a “fast on, fast off” approach that maintains high on-target editing while reducing cleavage at off-target sites, however OTEs can still occur. Several intelligently designed Cas9 variants have been described that reduce off-target activity but unfortunately also reduce on-target activity when used in RNP format. IDT has developed a novel mutant HiFi Cas9 protein that was evolved to reduce off-target gene editing while maintaining on-target potency that can be used as RNP. Methods to maximize editing specificity will be described as well as methods to optimize HDR efficiency.