To the Editor:
The clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas system is a highly efficient technology for genome editing of mouse zygotes1. Previously, we reported cotransmission of a Cas9-induced mutation in the X-linked Ar gene and an off-target mutation to offspring of founder animals from pronuclear injection of Cas9 mRNA and two single guide RNAs (sgRNAs)2. No off-target damage is observed in offspring derived from founder animals injected with Cas9 nickase mRNA and a pair of sgRNAs, showing that CRISPR-Cas specificity is improved by employing nickases2,3. Here, we used whole-genome sequencing to more thoroughly assess any damage induced by injection of Cas9 versus Cas9 nickase.
Two female founder animals (C57BL/6J × CBA) carrying biallelic deletions in Ar induced with Cas9 and Cas9 D10A nickase (animals F18 and F25, respectively) were mated to C57BL/6J males, and heterozygous Ar mutant offspring were sequenced. To control for strain-specific variants, we also sequenced a C57BL/6J and a CBA animal from our breeding colonies. Whole-genome sequencing was performed at a sufficient depth (20–25×) to detect more than 95% of heterozygous variants (Supplementary Methods).
We applied two standard computational methods to detect small insertions and deletions (indels), the hallmark of endonuclease-induced damage. We readily detected the Ar mutant alleles transmitted to each of the F1 offspring (Supplementary Fig. 1). We then looked for damage to related sites in the genome with up to five mismatches. Of 8,441 possible off-target sites (Table 1), damage was observed only in offspring of the Cas9-treated founder animal at OTS3, a bona fide off-target site for one of the two Ar sgRNAs2 (Supplementary Fig. 2).
We extended our analysis to the remainder of the genome on the basis of the following assumptions. We reasoned that the variants would not be shared with the control inbred strains (C57BL/6J or CBA) and would not be shared in offspring from individual founder animals. Variants associated with repetitive DNA sequence were discarded as these cannot be distinguished from the large number of naturally occurring variants in repetitive DNA sequence in individual mice. These filtering steps (Supplementary Tables 1 and 2) reduced the number of indels to a small set of 120 high-quality variants (Supplementary Data). Twenty-four variants were selected at random, of which 22 were confirmed by experimental validation.
On the basis of the ungapped and gapped alignments of the two Ar sgRNAs to genomic sequence at the variant sites, none of the 120 variants appeared to be a true off-target site (Supplementary Fig. 3). We conclude that these variants arose spontaneously and were not the result of unconstrained Cas9 endonuclease activity. Our study is consistent with three recent reports showing negligible genome-wide damage in Cas9-engineered human induced pluripotent stem cells4,5,6.
In contrast to embryonic stem cell technology, where extensive genetic variation arises in culture, undesired mutations induced by the Cas9 endonuclease will be rare in zygotes. Because unlinked mutations will segregate away through breeding, phenotyping of two independent founder animals would be sufficient to establish causality. Alternatively, mutant founder animals generated with two unrelated guide RNAs would rigorously control for any confounding alleles.
V.I. and B.S. contributed equally to this work. V.I., A.H. and T.K. performed the computational analysis of whole genome sequence. B.S. and W.Z. experimentally validated the variants in mice. X.H. and W.C.S. wrote the manuscript.
Wang, H. et al. Cell 153, 910–918 (2013).
Shen, B. et al. Nat. Methods 11, 399–402 (2014).
Ran, F.A. et al. Cell 154, 1380–1389 (2013).
Smith, C. et al. Cell Stem Cell 15, 12–13 (2014).
Veres, A. et al. Cell Stem Cell 15, 27–30 (2014).
Suzuki, K. et al. Cell Stem Cell 15, 31–36 (2014).
We thank the Sanger Institute sequencing pipeline for whole-genome sequencing. This work was supported by the 973 program (2011CB944301) to X.H. and a core grant from the Wellcome Trust to W.C.S.
The authors declare no competing financial interests.
Integrated supplementary information
The raw PINDEL output (top panel) and pileup of supporting sequence reads (lower panel) for F1 animal F18-9 shows a heterozygous deletion of 58 bp in exon 1 of Ar, previously characterized in founder animal F18 by PCR sequencing2. Note the fall in read coverage across the region and the mapping of soft-clipped reads.
The raw PINDEL output (top panel) and pile up of supporting reads (lower panel) for F1 animal F18-9 shows a heterozygous deletion of 2 bp in OTS3 (Chr8;:121017179-121017180), detected previously by PCR sequencing2.
Supplementary Figure 3 Alignment of Ar sgRNAs to high-quality variants do not correspond to a bona fide off-target site
The PAM site in the genomic sequence is highlighted in red text. (a) Ungapped alignment showing 7 mismatches between the sgRNA and genome sequence. The variant sequence is located 9 bp upstream of the predicted Cas9 cleavage site (arrowhead). The 2bp deletion detected at this site (Chr10:39516743) in animal F18-6 is underlined. (b) Gapped alignment showing 4 mismatches. The variant lies outside of the target sequence, 8 bp downstream of the predicted Cas9 cut site (arrowhead). The 6 bp deletion detected at this site (Chr3:141956378) in animals F25-5 and F25-6 is underlined.
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Iyer, V., Shen, B., Zhang, W. et al. Off-target mutations are rare in Cas9-modified mice. Nat Methods 12, 479 (2015). https://doi.org/10.1038/nmeth.3408
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