ArticleCell reports methods2026
Optimizing microhomology-based genome editing by engineering DNA polymerase θ for improved efficiency and reduced on-target indels.
Article in Cell reports methods, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Precise genome editing through targeted DNA insertion is critical for gene therapy and biomedical research. While existing methods rely on homology-directed repair (HDR), this process suffers from low efficiency in non-dividing cells. Microhomology-mediated repair provides an alternative but remains intrinsically inefficient. Here, we developed a DNA polymerase θ (Pol θ)-based editor (PET) to enhance kilobase-scale targeted DNA integration across diverse genomic loci and cell types. We demonstrate that polymerase domain (pPET) and helicase-like domain (hPET) independently improve microhomology-mediated editing, with pPET achieving a 3-fold increase in knock-in rates over conventional SpCas9-mediated editing. Next-generation sequencing reveals that pPET and hPET reduce on-target indel rates by ∼30%, while increasing precise insertions by up to 80%. Compared with other Pol θ domain configurations and MMEJ-enhancing factors, pPET exhibits superior performance. These findings establish Pol θ functional domains as effective tools for improving microhomology-driven genome editing and advancing therapeutic applications.
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