CRISPR Method of Gene Editing Creates Massive Genetic Reshuffling

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Drug developers relying on CRISPR-Cas9 platforms for the creation of gene therapies saw their shares slide when it was revealed that the technique may inadvertently cause disruptions of larger chunks
Drug developers relying on CRISPR-Cas9 platforms for the creation of gene therapies saw their shares slide when it was revealed that the technique may inadvertently cause disruptions of larger chunks

A lot is banking on the success of CRISPR-Cas9 as a gene-editing tool, and many companies have been created specifically on the anticipated success of the tool that harnesses a bacterial immune system.

However, another new paper has been released that may put a dent in the approach that was thought to be the basis of many new gene-editing therapies, many of which are being developed for the treatment of various cancers.1

Researchers from the Wellcome Sanger Institute revealed on July 16 in a study published in Nature Biotechnology that in addition to causing breaks in the DNA at target sites, CRISPR-Cas9 also causes unintended deletions of larger chunks of the genetic code, resulting in mutations a distance away from the DNA cleavage site.

In addition, many of these deletions and insertions happened at locations that would typically be missed by standard genotyping methods, as they occurred further away from the target site than was previously surveyed. Previous studies that attempted to assess the safety of the approach validated the extent of changes to the genome at regions close to the cleavage site.

According to corresponding author of the study, Allan Bradley, PhD, director emeritus of the Wellcome Sanger Institute and chief technical officer of Kymab, the changes CRISPR causes to the genome "have been seriously underestimated before now."2 As the research team wrote in the study, "The observed genomic damage in mitotically active cells caused by CRISPR­­­­­­­­­­-Cas9 editing may have pathogenic consequences."1

Cells lines taken from both mice and humans were examined for CRISPR-Cas9's effects, and researchers introduced the platform into cells via viral vectors as well as through transient transfection (to prove that the results they saw were not a result of issues with the vector model of delivery).

In the mouse models, they used long reads produced by PacBio sequencers to determine the extent of changes associated with knock-out of PigA. Coupled with results from Sanger sequencing, the researchers determined that there were lesions (such as large deletions or insertions) in 23 of the 133 recovered alleles.

Other lesions with inversions of the regions containing the exon were found as well. "Had the assessment been limited to the immediate vicinity of the cleavage site, such alleles would have been misclassified as wild type, and their phenotypic consequences would have been underestimated," the researchers wrote.

The researchers looked at human pigmented retinal epithelial cell lines to confirm that genomic damage was wide-ranging and was not restricted to the mouse cell line model. They found that in all the cell lines tested, mutations they observed could trigger new tumors.

The first clinical trials using CRISPR-Cas9 as a gene editing platform are currently underway, and the trials mentioned by the study researchers are all under investigation for therapies to treat cancer. Specifically, they are looking at CRISPR-modified therapies for the treatment of leukemia and lymphoma (ClinicalTrials.gov identifiers: NCT03398967 and NCT03166878), esophageal cancer (ClinicalTrials.gov identifier: NCT03081715), non-small cell lung cancer (ClinicalTrials.gov identifier: NCT02793856), Epstein-Barr virus-associated malignancies (ClinicalTrials.gov identifier: NCT03044743), and hematological malignancies in patients with HIV (ClinicalTrials.gov identifier: NCT03164135).

Older methods of editing the genome, including techniques such as transcription activator-like effector nucleases and zinc-finger nucleases, serve as alternatives to CRISPR.

References

  1. Kosicki M, Tomberg K, and Bradley A. Repair of CRISPR-Cas9-induced double-stranded breaks lead to large deletions and complex rearrangements [published online July 16, 2018]. Nat Biotechnol. doi: 10.1038/nbt.4192
  2. Wellcome Sanger Institute. Genome damage from CRISPR/Cas9 gene editing higher than thought [news release]. https://www.sanger.ac.uk/news/view/genome-damage-crisprcas9-gene-editing-higher-thought. Accessed July 19, 2018.

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