Researchers Report Fewer Large DNA Deletions After Base- And Prime Editing Compared to CRISPR-Cas9 Editing

In an article published this week in Nature Biomedical Engineering, researchers in Korea report that large DNA deletions (> 100 bp) occur during DNA repair at a much lower frequency for base editors and prime editors than for Cas9 nucleases (1).

The team performed in vitro gene editing on a panel of target sites in various cancer cell lines as well as human embryonic stem cells and human primary T cells from two donors. DNA deletions were detected using a combination of CRISPR-interference screening and an optimised long-range amplicon sequencing workflow that required the development of a k-mer alignment algorithm to simultaneously analyse large DNA deletions and small indels with high accuracy. The team found that Cas9-mediated double-stranded breaks induced large deletions at varying frequencies ranging from 0.2% to 17.5% across all six cell lines, excluding primary T cells, in which the frequency of large deletions was much higher (approx. 15 % averaged from both donors).

Further experiments with small molecule inhibitors of DNA repair pathways indicated that most of the deletions were the result of two DNA repair pathways: end resection and DNA-polymerase theta-mediated end joining. They also show that while base editors and prime editors generated large deletions despite the fact that they harbour mutated Cas9 nickases, the frequency of such deletions was about 20-fold lower after base- or prime editing than after CRISPR-Cas9 editing. The researchers highlight the importance of validating current gene-editing tools for clinical applications, and present their workflow as a strategy to evaluate unintended large or small changes to DNA arising from the application of these gene editors.

Read the full open-access article in Nature Biomedical Engineering here.