High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

Abstract

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

Fig. 1. Activities of RGENs harboring variant mismatched sgRNAs in a human cell-based EGFP disruption assay

(a) Schematic overview of the EGFP disruption assay. Repair of targeted Cas9-mediated double-stranded breaks in a single integrated EGFP-PEST reporter gene by error-prone NHEJ-mediated repair leads to frameshift mutations that disrupt the coding sequence and to associated loss of fluorescence in cells. Activities of RGENs harboring sgRNAs bearing (b) single mismatches, (c) adjacent double mismatches, (d) variably spaced double mismatches, and (e) increasing numbers of adjacent mismatches assayed on three different target sites in the EGFP reporter gene sequence. Mean activities of replicates (see Online Methods) are shown, normalized to the activity of a perfectly matched sgRNA. Error bars indicate standard errors of the mean. Positions mismatched in each sgRNA are highlighted in grey in the grid below. Sequences of the three EGFP target sites are shown in Supplementary Figure 2a.