Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing

Abstract

Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.

Figure 1. On-target and Off-target Mutations

(A) HUES 9 clones targeted in the SORT1 gene with TALENs or CRISPR-Cas9. (B) HUES 9 clones targeted in the LINC00116 gene with CRISPR-Cas9. For TALEN targeted clones, the boxes indicate the TALEN on-target and off-target binding sequences. For CRISPR-Cas9 targeted clones, the boxes indicate the 20-bp sequence matching the protospacer and the 3-bp PAM. For the on-target sites, deletions and insertions in the two alleles of each clone are indicated. For the off-target site, the mismatches with the TALEN on-target binding sequences are indicated in bold, and the deletion in one allele of the clone is indicated.