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

Ida Höijer,Rebecka Östlund,Anastasia Emmanouilidou,Selma Bozorgpana,Ulf Gyllensten,Lars Feuk,Marcel Den Hoed,Robin Van Schendel,Adam Ameur,Marcel Tijsterman

doi:10.1038/s41467-022-28244-5

Ida Höijer, Rebecka Östlund + Show 8 more

Open Access

https://doi.org/10.1038/s41467-022-28244-5

Copy DOI

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.

Highlights

CRISPR-Cas[9] genome editing has potential to cure diseases without current treatments, but therapies must be safe
In order to conclusively determine the effects of CRISPR-Cas[9] and their long-term consequences in vivo, a large number of samples needs to be followed through development and over generations, using a sensitive method for genome analysis
To select gRNAs for our experiments, we pre-screened 23 gRNAs with high in vivo on-target efficiency. These gRNAs target zebrafish orthologues of human genes in loci identified by genome-wide association studies (GWAS) for cardiometabolic risk factors and diseases[34,35,36] and have been used to examine the role of the candidate genes in cardiometabolic disorders[37,38]

Summary

Introduction

CRISPR-Cas[9] genome editing has potential to cure diseases without current treatments, but therapies must be safe. 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. Undesired outcomes of CRISPR-Cas[9] genome editing have been the subject of many investigations The conclusions from these studies have been somewhat conflicting, with adverse effects of CRISPR-Cas[9], i.e., larger on-target structural variants (SVs) and offtarget mutations, being reported in some cases[5,6,7,8] but not in others[9,10]. The Nano-OTS method does not suffer from amplification bias, and reliably identifies off-target sites, even in repetitive and complex regions of the genome

Objectives

Methods

Results

Conclusion