CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences

TLDR

CRISPR/Cas9 is a versatile genome‑editing tool that efficiently targets DNA sequences complementary to its guide RNA, yet it can also cleave genomic sites containing mismatches to the guide strand. A better understanding of CRISPR/Cas9 specificity is needed to minimize off‑target cleavage in large mammalian genomes. Variants of single‑guide RNAs for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1‑ to 5‑bp bulges. We demonstrate that CRISPR/Cas9 can cleave genomic sites containing DNA or RNA bulges, that nickase‑based paired nicking tolerates bulges in one guide, that 15 of 114 predicted off‑target loci with a single‑base bulge and one to three mismatches were confirmed, and that comprehensive off‑target analysis including bulges is essential with guidelines to reduce potential cleavage.

Abstract

CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.