TWN Info Service on Biosafety
30 November 2021
Third World Network
www.twn.my
Dear Friends and Colleagues
Large Unintended Structural Changes Induced by CRISPR-CAS9 at Off-Target Sites in Fish
A study using CRISPR/Cas on zebrafish (Item 1) is the first to detect large unintended structural changes at off-target sites. The scientists examined the genome of zebrafish after using CRISPR/Cas and found that large structural changes (i.e. large insertions and deletions) had occurred at both the target sequence and at so-called off-target sites. Until now, it was unclear whether such large structural changes as described at on-target sites could also occur at off-target sites (Item 2).
The scientists had injected the gene scissors into zebrafish eggs, and subsequently examined both the genome of the fish that developed from the eggs and their offspring using modern sequencing methods. The examination of possible off-target sites revealed that unintended changes had occurred at three of them. Their analysis showed that mainly small changes, such as point mutations, were induced. However, larger changes were also observed: at one off-target site, for example, 903 base pairs of the DNA were deleted, thus changing a large part of a gene that was not supposed to be changed at all. The major structural changes at on- and off-target regions were found both in the fish that developed from the injected eggs and their offspring.
The results are highly relevant for clinical applications of CRISPR/Cas to avoid unexpected effects with major consequences for patients. In plant applications, however, no one has yet investigated whether large structural changes occur at off-target sites. Overall, it is important that the genome of the target organisms at on- and off-target sites is examined in detail after CRISPR/Cas experiments to detect unintended changes that may affect other gene regions.
With best wishes,
Third World Network
__________________________________________________________________________
Item 1
CRISPR-CAS9 INDUCES LARGE STRUCTURAL VARIANTS AT ON-TARGET AND OFF-TARGET SITES IN VIVO THAT SEGREGATE ACROSS GENERATIONS
Ida Höijer et al.
BioRxiv
doi: https://doi.org/10.1101/2021.10.05.463186
https://www.biorxiv.org/content/10.1101/2021.10.05.463186v1
5 October 2021
Abstract
To investigate the extent and distribution of unintended mutations induced by CRISPR-Cas9 in vivo, we edited the genome of fertilized zebrafish eggs and investigated DNA from >1100 larvae, juvenile and adult fish in the F0 and F1 generations. Four guide RNAs (gRNAs) were used, selected from 23 gRNAs with high on-target efficiency in vivo in previous functional experiments. CRISPR-Cas9 outcomes were analyzed by long-read sequencing of on-target sites and off-target sites detected in vitro. In founder larvae, on-target editing of the four gRNAs was 93-97% efficient, and three sites across two gRNAs were identified with in vivo off-target editing. Seven percent of the CRISPR-Cas9 editing outcomes correspond to structural variants (SVs), i.e., insertions and deletions ≥50 bp. The adult founder fish displayed a mosaic pattern of editing events in somatic and germ cells. The F1 generation contained high levels of genome editing, with all alleles of 46 examined F1 juvenile fish affected by on-target mutations, including four cases of SVs. In addition, 26% of the juvenile F1 fish (n=12) carried off-target mutations. These CRISPR-induced off-target mutations in F1 fish were successfully validated in pooled larvae from the same founder parents. In conclusion, we demonstrate that large SVs and off-target mutations can be introduced in vivo and passed through the germline to the F1 generation. The results have important consequences for the use of CRISPR-Cas9 in clinical applications, where pre-testing for off-target activity and SVs on patient material is advisable to reduce the risk of unanticipated effects with potentially large implications.
———————————————————————————————
Item 2
CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations
Project Genetic Engineering and the Environment
October 2021
Basics about changes to off-target and on-target sites
It is known from previous studies on mice and human cell lines that the use of CRISPR/Cas gene scissors can cause unintended changes in DNA at off-target sites. Off-target sites are very similar to the target sequence, which is why the gene scissors can also cut and induce alterations at these sites. Until now, only small changes, such as point mutations or short insertions and deletions (InDels) at off-target sites, have been observed. These small changes can also occur at the target sequence, sometimes called the on-target site. Larger structural changes have also been found at these locations, e.g. deletions of large parts of DNA sequences or newly inserted DNA sequences.
Results of the Höijer et al. study
Until now, it was unclear whether such large structural changes as described at on-target sites could also occur at off-target sites. The scientists from the present study tested this in zebrafish. They firstly analysed the genome of unmodified fish using modern sequencing methods in order to develop suitable guides (also called guide RNAs or gRNAs) for the gene scissors. Several different gRNAs were used. The overall aim of their experiments was to investigate possible structural changes at off-target sites, they therefore used gRNAs with the gene scissors that were very likely to cut at those sites. This allowed them to analyse changes caused at these DNA regions in more detail.
In various experiments, the scientists injected the gene scissors as a protein complex into zebrafish eggs, and examined both the fish that had developed from the eggs and their offspring using sequencing methods. It was found that the efficiency with which changes are obtained at the target sequence is high overall. Predominantly small mutations and, to a lesser extent, larger structural changes (large InDels) were induced.
Investigation of the DNA sequence at possible off-target sites revealed that unintended alterations had occurred at three of them. Their analysis showed that mainly small changes such as point mutations had been induced. However, they also observed larger changes in the DNA sequence. At one off-target site, for example, 903 base pairs (i.e. the letters of the DNA) were deleted, thus affecting a large part of a gene that was not supposed to be changed.
The major structural changes at on- and off-target sites were found in both the founder animals and their offspring.
Relevance of the results
The results are highly relevant for clinical applications of CRISPR/Cas to avoid unexpected effects with major consequences for patients. In plant applications, however, no one has yet investigated whether large structural changes occur at off-target sites. However, it is likely, as it has also been reported at on-target sites.
The design of CRISPR/Cas experiments is crucial for reducing unintended alterations at off-target sites. For example, this requires an appropriate gRNA design so that as few as possible off-target sites are affected, and a well-tuned concentration of the gene scissors used.
Overall, it is important that the genome of the target organisms at on- and off-target sites is examined in detail after CRISPR/Cas experiments to detect unintended changes that may affect other gene regions.
References
Höijer, I.; Emmanouilidou, A.; Östlund, R.; van Schendel, R.; Bozorgpana, S.; Feuk, L.; Gyllensten, U.; den Hoed, M.; Ameur, A. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. bioRxiv 2021, 2021.2010.2005.463186, doi:10.1101/2021.10.05.463186.