A group of researchers has shown that the DNA mismatch repair process plays a critical role in prime editing.
While CRISPR-Cas9-based genome editing technologies are powerful tools, the efficiency of current protocols is limited. The process also gives rise to undesirable effects that hinder their clinical implementation. To overcome some of the challenges, it is important to further explore the DNA pathways and mechanisms that impact editing outcomes.
Alternative editing approaches include base editing (BE) and prime editing (PE). BE involves nucleobase modification chemistry to incorporate single nucleotide variants. However, its use is limited to single-base substitutions. Meanwhile, PE is a highly versatile approach that allows for the replacement, insertion and deletion of indels and/or point mutations into any given genomic locus. This approach uses a combination of a Cas9(H840A) nickase1 and reverse transcriptase (RT) that is targeted to a precise genomic region by a PE guide RNA (pegRNA). Nonetheless, to date, the efficiency of prime editing has been highly variable across different genetic backgrounds, even within the same genomic locus and using the same pegRNA.
Identifying repair factors involve in prime editing
In a recent study, published in Nature Communications, researchers explored which factors influenced the success of PE by taking a closer look at DNA repair processes. To date, the underlying DNA repair machinery involved in PE is largely unknown. To address this, the researchers performed a targeted genetic screen for 32 DNA repair factors covering all known repair pathways.
Here, the team uncovered an inhibitory role for the mismatch repair (MMR) pathways in prime editing. They also showed that MMR proteins, MLH1 and MSH2, localised to sites of PE to directly counteract editing. In addition, the researchers found that deletion or transient depletion of these MMR factors increased PE efficiency 2 to 17-fold across different edit types, loci and cell lines.
Overall, these results provide new insights into the mechanisms of PE and suggest an approach that may help optimise its efficiency.
Study lead, Joanna Loizou, said:
“By removing the activity of the mismatch repair pathway from a cell, we show that the efficiency of prime editing can be increased, and its accuracy improved.”
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