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Background <p>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.</p> Method <p>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.</p> Results <p>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.</p> Conclusion <p>We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.</p> Graphical Abstract <p></p>

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Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月3日

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  • Daisuke Matsumoto,
  • Komari Kubota,
  • Yu Sato,
  • Tomoko Kato-Inui,
  • Kiyomi Nigorikawa,
  • Yuichiro Miyaoka,
  • Wataru Nomura

摘要

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