CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations.

Höijer I, Emmanouilidou A, Östlund R, van Schendel R, Bozorgpana S, Tijsterman M, Feuk L, Gyllensten U, den Hoed M, Ameur A

Nat Commun 13 (1) 627 [2022-02-02; online 2022-02-02]

CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.

Bioinformatics Support for Computational Resources [Service]

NGI Long read [Technology development]

NGI Uppsala (Uppsala Genome Center) [Technology development]

National Genomics Infrastructure [Technology development]

PubMed 35110541

DOI 10.1038/s41467-022-28244-5

Crossref 10.1038/s41467-022-28244-5

pmc: PMC8810904
pii: 10.1038/s41467-022-28244-5