Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin
Daisuke Matsumoto1,2,3, Komari Kubota4, Yu Sato5
1School of Pharmaceutical Sciences, Hiroshima University, 1-2-3 Kasumi Minami-Ku, Hiroshima, 734-8553, Japan. matsumoto-ds@igakuken.or.jp.
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.
