Supplementary data for "CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo"

Ida Höijer - Uppsala University, Science for Life Laboratory, SciLifeLab / Department of Immunology, Genetics and Pathology

Anastasia Emmanouilidou - Uppsala University, Science for Life Laboratory, SciLifeLab / Department of Immunology, Genetics and Pathology

Rebecka Östlund - Uppsala University, Department of Immunology, Genetics and Pathology

Robin van Schendel - Leiden University Medical Center, Department of Human Genetics

Lars Feuk - Uppsala University, Science for Life Laboratory, SciLifeLab / Department of Immunology, Genetics and Pathology / Department of Women's and Children's Health, Research group (Dept. of women´s and children´s health)

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.

The data files contain supplementary information for the manuscript "CRISPR-Cas9 induces large structural variant