A new Nature Biotechnology publication is sending reverberations through the CRISPR and gene therapy communities. The discovery that the widely used CRISPR/Cas9 method results in far more genomic changes than previously thought — including big deletions and rearrangements — was made possible by the use of long-read SMRT Sequencing.
“Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements” comes from Michael Kosicki, Kärt Tomberg, and Allan Bradley at the Wellcome Sanger Institute. The scientists aimed to better understand the possible universe of on-target edits (rather than the better-studied off-target effects) made in a controlled environment, starting with a 5.7 kb amplicon from the X-linked PigA locus in mouse embryonic stem cells. “Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific,” they write.
Their findings led to a collective groan among CRISPR scientists and the businesses based on this technology. “We report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line,” Kosicki et al. report. “We speculate that current assessments may have missed a substantial proportion of potential genotypes generated by on-target Cas9 cutting and repair, some of which may have potential pathogenic consequences following somatic editing of large populations of mitotically active cells.”
The heterogeneous nature of DNA repair after CRISPR edits was previously observed by Gasperini et al. which shared the strategy of long read SMRT sequencing to get a more clear picture of editing outcomes. In both cases, choosing long-read SMRT Sequencing allowed a larger region adjacent to the intended edit site to be surveyed, uncovering unexpected changes caused by CRISPR-Cas9 cuts. A number of these changes would have been impossible to spot with short-read sequencing, such as large edits deleting an adjacent primer binding site that would have been used to check the region. “The most frequent lesions in these cells were deletions extending many kilobases up- or downstream, away from the exon,” the scientists note. “We conclude that, in most cases, loss of PigA expression was likely caused by loss of the exon, rather than damage to intronic regulatory elements.” In one case, the team even found a de novo insertion — “a perfect match to four consecutive exons derived from the Hmgn1 gene” — that they believe came from spliced, reverse-transcribed RNA.
These sweeping edits weren’t the only bad news in the paper. The scientists repeated the original experiment four times to determine whether the same edits would be seen each time and found that they were not. “Each biological replicate differed substantially, despite a large number of unique deletion events sampled, indicating that the diversity of potential deletion outcomes is vast,” they report.
The CRISPR method has been considered quite promising as a gene-editing tool to cure disease, and this publication does not suggest that the authors’ findings would necessarily derail that idea. Instead, they urge others in the field to be more comprehensive in analyzing genomes before and after the use of CRISPR for a clearer view of its effects. “Results reported here … illustrate a need to thoroughly examine the genome when editing is conducted ex vivo,” they conclude. “As genetic damage is frequent, extensive and undetectable by the short-range PCR assays that are commonly used, comprehensive genomic analysis is warranted to identify cells with normal genomes before patient administration.”