More trouble than expected

A Wellcome Sanger Institute team has revealed that CRISPR/Cas9 editing can result in more damage to the genome than previously believed

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CAMBRIDGE, U.K.—The CRISPR/Cas9 gene-editing approach has been the focal point of a great deal of interest and research of late, as many wonder if this could be the answer to one day correcting genetic diseases. But others have just as loudly argued that further research needs to be done, and it looks as though they might be right to recommend caution—a new study out of the Wellcome Sanger Institute has showed that CRISPR/Cas9 editing can result in genetic damage that is more extensive than anticipated. Authors Michael Kosicki, Kärt Tomberg and Prof. Allan Bradley, all of the Wellcome Sanger Institute, shared their results in a paper titled “Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.” This study was published in Nature Biotechnology.
CRISPR/Cas9 works by cutting DNA at certain points along the strand and introducing changes at those points, such as by removing miscoded regions or inserting properly coded sections that might be missing. Previously, such gene editing hadn't resulted in a lot of unexpected mutations at the editing site, but the issue, as noted in the Nature Biotechnology paper's abstract, is that “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.” When the Wellcome Sanger Institute team looked deeper in a full systematic study, they found significant mutations, some of them too far from the editing site to have been detected with standard genotyping methods.
The authors note in their paper that “The vast majority of on-target DNA repair outcomes after Cas9 cutting in a variety of cell types are thought to be insertions and deletions (indels) of less than 20 bp13,14,15. Although indels a few hundred nucleotides in size were also observed in experiments using Cas9 or other nucleases, they were reported to be rare16,17,18. Consequently, Cas9 has been assumed to be reasonably specific and the first approved clinical trials using Cas9 edited cells are underway.”
In their experiments, however, they found “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. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.”
“This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects,” said Bradley, corresponding author on the study.
Bradley, Kosicki and Tomberg noted that their work “was conducted at actively transcribed loci in normal ES cells and progenitor cells, both with intact DNA repair processes, as well as in an immortalized, differentiated human cell line; each are surrogates for various clinical editing applications. We show that extensive on-target genomic damage is a common outcome at all loci and in all cell lines tested.”
“Moreover, the genetic consequences observed are not limited to the target locus, as events such as loss-of-heterozygosity will uncover recessive alleles, whereas translocations, inversions and deletions will elicit long-range transcriptional consequences,” the authors explain. “Given that a target locus would presumably be transcriptionally active, mutations that juxtapose this to one of the hundreds of cancer-driver genes may initiate neoplasia. In the clinical context of editing many billions of cells, the multitude of different mutations generated makes it likely that one or more edited cells in each protocol would be endowed with an important pathogenic lesion. Such lesions may constitute a first carcinogenic 'hit' in stem cells and progenitors, which have a long replicative lifespan and may become neoplastic with time. Such a circumstance would be similar to the activation of LMO2 by pro-viral insertion in some of the early gene-therapy trials, which caused cancer in these patients30.
“Results reported here also illustrate a need to thoroughly examine the genome when editing is conducted ex vivo. 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.”
Some efforts are underway at other organizations to try improve on current methods of gene editing, and these results from the Wellcome Sanger team will likely only further bolster those efforts. As covered in a recent piece by our Chief Editor Jeffrey Bouley, researchers at the University of Texas at Austin are exploring the potential of using the Cas12a protein instead, which they found to be more effective and precise than Cas9. Given the promise of genome editing, it's not surprising that the roadblocks encountered so far have only inspired scientists to find workarounds that could make gene editing a reality.

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