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A Helicase-Fused Cas9 improves Large-Size Fragment Knock-in
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DOI：10.1016/j.ymthe.2026.07.049.png)
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Abstract
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Homologous recombination (HR) mediated large-size fragment knock-in (ls-KI) remains inefficient in mammalian cells, even with the assistance of CRISPR/Cas9. We hypothesized that adding DNA helicase activity to CRISPR/Cas9 could enhance ls-KI efficiency. To test this, we fused MCM5, a subunit of the eukaryotic MCM2-7 helicase complex, to the N-terminus of spCas9. The resulting fusion protein, termed MCCas, markedly increased knock-in rates across multiple loci in different human cell lines, significantly outperforming spCas9. Remarkably, MCCas enabled efficient ls-KI with donor templates of up to 10 kb in size. MCCas mediated ls-KI, in comparison to those mediated by spCas9, is also associated with reduced frequencies of on-target and off-target insertion and deletion (indel) events. Mechanistic investigations revealed that MCCas-mediated ls-KI relies on the canonical HR pathway, as inhibition of key processes such as end resection and strand invasion abolished the enhancement. To further validate its application, we employed MCCas to knock in the human ACE2 (hACE2) coding sequence to the rabbit genome. Consistent with our findings in human cells, MCCas led to more than two-fold increase in ls-KI rates in rabbit embryos compared to spCas9. Collectively, our results establish MCCas as a promising gene editing tool with enhanced ls-KI capacity.
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