The CRISPR–Cas9 system is thought to be a reasonably specific method for genome editing. But Bradley and colleagues now report that CRISPR–Cas9 induces extensive on-target mutagenesis in mouse and human cells, calling for greater caution when using it in clinical contexts and stressing the need for comprehensive genomic analyses before edited cells can be used in patients.
The authors analysed the allelic diversity induced by the introduction of Cas9 and single-guide RNAs that target introns and exons of PigA (an X-linked locus) in mouse embryonic stem cells (ESCs), as targeting the introns also led to the loss of PigA. Sequencing of PigA-deficient single-cell clones revealed that, in addition to creating the expected insertions and deletions (indels) of <50 bp, CRISPR–Cas9 generated deletions of >250 bp to 6 kb in more than 20% of alleles. Moreover, single-nucleotide polymorphisms, indels and large deletions and insertions that were non-contiguous with the Cas9 cut site were identified; such mutations would be missed if the analysis was limited to the vicinity of the cleavage site, as is often the case. In some cases, changes in parts of the genome distant from the target site were also found.
By editing and analysing other loci in different cell lines — immortalized human retinal pigment epithelial cells and progenitor cells isolated from mouse bone marrow — the authors confirmed that the extensive on-target damage, which is associated with DNA repair, is not an intrinsic property of mouse ESCs.
Thus, CRISPR–Cas9 can induce extensive on-target genomic rearrangements that could potentially lead to pathogenic lesions in clinically relevant cell types.
References
Original article
Kosicki, M. et al. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018)
Further reading
Dominguez, A. A. et al. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016)
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Baumann, K. Not so CRISP(R). Nat Rev Mol Cell Biol 19, 619 (2018). https://doi.org/10.1038/s41580-018-0056-4
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DOI: https://doi.org/10.1038/s41580-018-0056-4