Kosicki M, Tomberg K, Bradley A.
Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.
Nat Biotechnol. 2018 Jul 16;
PubMed.
This data from Allan Bradley and colleagues suggest there could be problems using CRISPRs in dividing cells, because the authors found large deletions and more complex genomic rearrangements at targeted sites. Though it has been long recognized that unwanted editing could be an issue with CRISPR/Cas9, the extent of this effect, as seen in this paper, is surprising. One thing to note is that each intervention (i.e. guide RNA/Cas9 pairs) will likely have its own repertoire of unwanted editing, and it’s possible that the gRNAs used in this paper are particularly problematic in this regard. I am not sure what the standard is for looking at DNA damage in patients in CRISPR trials, but this is obviously an extremely important parameter to keep in mind. In other words, we need to have a reasonably good idea of what the rate and extent of unwanted editing would be, before therapies can be initiated. This would obviously be even more problematic in the case of neuronal diseases, as—unlike hematopoietic cells—it will not be easy to do careful analyses on cells in the brain.
Genome editing is a potentially powerful approach for knocking out disease-causing genes, repairing genes whose mutations have resulted in a loss of function, or safely inserting therapeutic genes into the host cell genome. It is very important to investigate all consequences that can occur to the genome when using a nuclease such as Cas9 to introduce a DNA break. This manuscript reports important side effects of introducing a double-stranded break, namely that it can result in larger-than-anticipated deletions at or near the cut site, or even insertion of exogenous DNA at that site.
The authors are to be commended for adding this important information to the literature, and it must be further investigated and considered, especially for therapeutic applications of genome editing. Fortunately, it is possible to stimulate the repair or insertion of genes by methods other than using a double-stranded breakage (DSB), instead introducing a single-stranded nick. Nicks did not appear to be analyzed in this study, but they would likely result in less damage. For knocking out a disease-causing gene, DSB-induced deletions that are larger than intended may be acceptable, since the goal is ablating expression of the gene altogether. That said, further investigation will hopefully provide quantitative insights into the frequency of other longer-range consequences of a DSB, such as translocations or large scale deletions, which would represent a safety concern.
Comments
University of California, San Diego
This data from Allan Bradley and colleagues suggest there could be problems using CRISPRs in dividing cells, because the authors found large deletions and more complex genomic rearrangements at targeted sites. Though it has been long recognized that unwanted editing could be an issue with CRISPR/Cas9, the extent of this effect, as seen in this paper, is surprising. One thing to note is that each intervention (i.e. guide RNA/Cas9 pairs) will likely have its own repertoire of unwanted editing, and it’s possible that the gRNAs used in this paper are particularly problematic in this regard. I am not sure what the standard is for looking at DNA damage in patients in CRISPR trials, but this is obviously an extremely important parameter to keep in mind. In other words, we need to have a reasonably good idea of what the rate and extent of unwanted editing would be, before therapies can be initiated. This would obviously be even more problematic in the case of neuronal diseases, as—unlike hematopoietic cells—it will not be easy to do careful analyses on cells in the brain.
View all comments by Subhojit RoyInstitute for Multigenerational Studies
Genome editing is a potentially powerful approach for knocking out disease-causing genes, repairing genes whose mutations have resulted in a loss of function, or safely inserting therapeutic genes into the host cell genome. It is very important to investigate all consequences that can occur to the genome when using a nuclease such as Cas9 to introduce a DNA break. This manuscript reports important side effects of introducing a double-stranded break, namely that it can result in larger-than-anticipated deletions at or near the cut site, or even insertion of exogenous DNA at that site.
The authors are to be commended for adding this important information to the literature, and it must be further investigated and considered, especially for therapeutic applications of genome editing. Fortunately, it is possible to stimulate the repair or insertion of genes by methods other than using a double-stranded breakage (DSB), instead introducing a single-stranded nick. Nicks did not appear to be analyzed in this study, but they would likely result in less damage. For knocking out a disease-causing gene, DSB-induced deletions that are larger than intended may be acceptable, since the goal is ablating expression of the gene altogether. That said, further investigation will hopefully provide quantitative insights into the frequency of other longer-range consequences of a DSB, such as translocations or large scale deletions, which would represent a safety concern.
View all comments by J. David SchafferMake a Comment
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