利用EvolvR系统对酶基因实现多窗口定向进化的研究

EvolvR系统是一种CRISPR介导的新型定向进化技术,本研究探索其在酶基因序列定向进化中的适用性以及突变效率,同时在此基础上扩大EvolvR的突变窗口长度.研究成功将4个能高效表达的单个sgRNA串联,并检测到靶向同一基因的3个不同靶位点.证明EvolvR具有在目标基因区域大范围制造突变的潜力,具有很高的应用价值....

Research on the multi windows directed evolution of target enzyme gene using EvolvR system

Directed evolution includes generation of random gene library, expression of genes in a suitable host and selection of target variants. EvolvR is a new CRISPR-mediated directed evolution technology that allows target sequence to evolve in one day. At present, EvolvR only has been used in antibiotic resistance genes, and attempts to connect two sgRNAs at most to expand the length of the mutation window. This study aims to explore the applicability and mutation efficiency of the EvolvR system in the directed evolution of enzymes and the length of the mutation window. The results showed that concatenation of four efficiently expressed individual sgRNA could indeed target three sites of a same gene. EvolvR was proved to have the potential to produce mutations in a wide range of target gene regions and has high application value. This study laid a foundation for the targeted enzyme directed evolution with EvolvR system.