Low levels of off-target effects in gene-editing of animal zygotes using CRISPR method

A follow up to the previous posts, with further confirmation that off-target changes in gene-editing can be minimised

 Off-target effect in complete knockout animals by C-CRISPR

Finally, we examined whether multiple-sgRNA targeting by the C-CRISPR method induces more off-target effects than single-sgRNA targeting. As previously described22, we examined up to 10 off-target sites for each sgRNA in six mice derived from a 4-sgRNA-targeting mutagenesis (Tyr-B+C+D+E-#1, #2, #3, #4, #5, #6), and two monkeys from 3-sgRNA targeting (Prrt2-B+C+D-#11, #12). DNA sequencing of the PCR products amplified from these genomic sites showed that no mutations occurred at any of these loci (Supplementary information, Figure S3A and S3B). We also performed whole-genome sequencing on these samples, as well as on two control monkeys from single-sgRNA targeting (Prrt2-A-#8, #9) at a sufficient depth to detect off-target mutations (15 × for the mice and 25 × for the monkeys).

We then searched for off-target effects in the genome allowing up to five mismatches of 4 sgRNAs in the mice (Tyr-B, C, D, and E) and 3 sgRNAs (Prrt2-B, C, and D) in the monkeys (Table 1; Supplementary information, Table S2 and S3). Of 10 201 and 19 447 possible off-target sites in mice and monkeys, respectively, we found no indels in the six mice and monkey #12 after filtering variants shared in individual samples as done in previous studies23,24 (Table 1; Supplementary information, Table S3). For monkey #11, we observed indels in three off-target sites, with five mismatches in both PAM-distal and PAM-proximal regions (Supplementary information, Table S3).

We further examined the three off-target sites in monkey #11 by PCR amplification and sequencing, and found that one site was not a true off-target site (Supplementary information, Figure S3C and S3D) and the other two sites were associated with a repeated DNA sequence, which could not be distinguished by sequencing (Supplementary information, Table S3).

We also examined genomic rearrangements, including deletions, duplications, inversions, and copy number variations, using the similar strategies, and found no rearrangements in the six mice and two monkeys (#11 and #12) (Supplementary information, Table S3). Thus C-CRISPR approach did not induce significant off-target alterations in gene-edited mice and monkeys beyond that expected for CRISPR/Cas9-mediated editing in general1,24,25,26.


From Cell Research - One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs: