IS200/IS605转座子家族编码的核酸酶OgeuIscB基因编辑体系的优化

Optimization of the gene editing system for nuclease OgeuIscB encoded by the IS200/IS605 transposon family

目的提升来源于IS200/IS605转座子家族的RNA引导的核酸内切酶OgeuIscB的基因编辑效率。方法利用OgeuIscB蛋白已解析的晶体结构,计算氨基酸与核酸之间的原子距离,将距离小于10的非正电荷氨基酸残基突变为精氨酸(arginine,R)以提高OgeuIscB与核酸的亲和力,通过人胚肾293T细胞内源性位点验证不同突变体的基因编辑效率。结果经过两轮迭代突变,发现Q376R-S456R和A401R-S456R这2个双突变体在不同细胞系的多个内源性位点上均能显著提升OgeuIscB的基因编辑效率。结论基于结构对OgeuIscB蛋白进行理性蛋白质工程化改造,能够提升OgeuIscB核酸内切酶活性,为将其作为一种有效的基因编辑工具提供了实验依据。

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 10 were mutated into arginine(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 improve the activity of OgeuIscB endonuclease,providing experimental evidence for its use as an effective gene editing tool.