PUBLICATION
CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations
- Authors
- Höijer, I., Emmanouilidou, A., Östlund, R., van Schendel, R., Bozorgpana, S., Tijsterman, M., Feuk, L., Gyllensten, U., den Hoed, M., Ameur, A.
- ID
- ZDB-PUB-220204-2
- Date
- 2022
- Source
- Nature communications 13: 627 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- DNA
- Gene Editing/methods*
- Genetic Therapy
- Germ Cells
- Humans
- Mutation
- RNA, Guide, Kinetoplastida/genetics
- Zebrafish/genetics*
- PubMed
- 35110541 Full text @ Nat. Commun.
Citation
Höijer, I., Emmanouilidou, A., Östlund, R., van Schendel, R., Bozorgpana, S., Tijsterman, M., Feuk, L., Gyllensten, U., den Hoed, M., Ameur, A. (2022) CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nature communications. 13:627.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping