利用EvolvR系统对酶基因实现多窗口定向进化的研究

定向进化包括随机基因文库生成、基因在合适宿主中的表达和筛选目标特性变体. EvolvR作为一种CRISPR介导的新型定向进化技术，可以使目标序列在一天内完成整个进化过程. 目前，EvolvR只报导用于抗性基因的突变，以及串联最多2个sgRNAs以扩大突变窗口长度. 本研究旨在探究EvolvR系统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. 该研究结果表明，将4个能高效表达的单个sgRNA 串联，成功检测到靶向同一基因的3个不同靶位点，证明了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. 本研究为利用EvolvR系统对目标酶基因实现定向进化的研究奠定了基础.

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.