Abstract Background In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies. Method In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect. Results By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9. Conclusion We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system. Graphical Abstract
Tomoko Kato-Inui (2025) studied this question.