Optimization of the gene editing system for nuclease OgeuIscB encoded by the IS200/IS605 transposon family
Objective To improve the gene editing efficiency of the RNA-guided endonuclease OgeuIscB derived from the IS200/IS605 transposon family.Methods Using the resolved crystal structure of OgeuIscB protein,the atomic distance between amino acids and nucleic acids was calculated.The non-positively charged amino acid residues with a distance of less than 10Åwere mutated into argi-nine(R)to improve the affinity between OgeuIscB and nucleic acids.The gene editing efficiency of different mutants was verified by the endogenous sites in human embryonic kidney 293T cells.Results After two rounds of iterative mutations,it was found that both Q376R-S456R and A401R-S456R double mutants significantly improved the editing efficiency of OgeuIscB at multiple endogenous sites in different cell lines.Conclusion The rational protein engineering modification of OgeuIscB protein based on its structure can im-prove the activity of OgeuIscB endonuclease,providing experimental evidence for its use as an effective gene editing tool.
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