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

Ida Höijer (), Anastasia Emmanouilidou, Rebecka Östlund, Robin Schendel, Selma Bozorgpana, Marcel Tijsterman, Lars Feuk, Ulf Gyllensten, Marcel Hoed and Adam Ameur ()
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Ida Höijer: Uppsala University
Anastasia Emmanouilidou: Uppsala University
Rebecka Östlund: Uppsala University
Robin Schendel: Leiden University Medical Center
Selma Bozorgpana: Uppsala University
Marcel Tijsterman: Leiden University Medical Center
Lars Feuk: Uppsala University
Ulf Gyllensten: Uppsala University
Marcel Hoed: Uppsala University
Adam Ameur: Uppsala University

Nature Communications, 2022, vol. 13, issue 1, 1-10

Abstract: Abstract 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.

Date: 2022
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DOI: 10.1038/s41467-022-28244-5

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