基于链霉菌I-E型CRISPR-Cas系统的新型基因编辑工具的开发与应用

The popular genome editing tool CRISPR-Cas9, derived from Streptococcus pyogenes, now has been questioned about the off-target problems. Streptomyces avermitilis, a producer of important pesticide avermectin, own an active and high specific type I-E CRISPR-Cas system, which may be able to edit genome. Type I-E system in S. avermitilis is highly specific. It can degrade foreign plasmids containing protospacer completely corresponding to spacer, rather than plasmids containing protospacer with mismatches, strongly suggesting that the system in S. avermitilis can be developed as a high specific genome editing tool which can avoid off-target problems. And we found that type I-E systems exist in 17 Streptomyces strains with similarity of the system in S. avermitilis. But the functions and features of these systems are still unclear.. In this project, we will identify the functions and features of the type I-E systems in 17 Streptomyces strains. And then, we will design gene editing tools derived from active type I-E system in Streptomyces to edit genome of Streptomyces itself. Firstly, an artificial CRISPR array corresponding to target gene will be designed and constructed into a plasmid to generate a gene editing tool. Then the tool will be applied to implement gene knockout, gene cluster knockout, gene mutation and multiple gene knockout simultaneously. After that the properties of the tool will be valued and the tool will be optimized. . More than that, we will apply active type I-E system in Streptomyces to edit genome in heterologous cells including Streptomyces and mammalian cells. Genes casABCDE and cas3 will be codon optimized and cloned into a Streptomyces or eukaryote expression vector. Meanwhile, an artificial CRISPR array corresponding to target gene will be designed and constructed into the vector to generate a genome editing tool. The efficiency of the tool in heterologous cells will be valued.. After all, we may deliver a type I-E system based gene editing tool derived from Streptomyces. It may can be used as a high specific gene editing tool avoiding off-target problems that CRISPR-Cas9 system is involved in.

CRISPR-Cas9基因组编辑技术存在的脱靶问题严重制约了其在临床上的应用，提高其特异性或寻找新的高特异性基因编辑工具是主要的解决途径。申请人前期工作证明阿维链霉菌中存在活跃的I-E型CRISPR-Cas系统，该系统特异性高，仅降解与Guide RNA完全互补的核酸序列，有望解决CRISPR-Cas9系统的脱靶问题。此外，申请人还发现17株链霉菌中存在与阿维链霉菌类似的I-E型CRISPR-Cas系统，其功能和特性还未鉴定，编辑基因的能力还有待证明。基于此，本项目将鉴定和分析该17株链霉菌中的I-E型CRISPR-Cas系统，并尝试以之建立新的基因编辑工具，用于链霉菌自身基因和哺乳动物细胞基因的编辑，有望开发出一种高特异性且具有自主知识产权的基因编辑新工具。

CRISPR-Cas9系统在链霉菌等多个物种的基因组编辑中得到了广泛应用，但其普遍存在的脱靶问题仍未得到有效解决。阿维链霉菌中存在活跃的I-E型CRISPR-Cas系统，可能具有与CRISPR-Cas9系统类似的编辑基因的能力，有被开发为新的基因编辑工具的潜能。且该系统特异性高，仅降解与Guide RNA完全互补的核酸序列，有望解决CRISPR-Cas9系统的脱靶问题。此外，链霉菌中广泛存在与阿维链霉菌类似的I-E型系统，其功能和特性还未鉴定，编辑基因的能力还有待证明。基于此，本项目首先利用生物信息学技术分析鉴定17株链霉菌中的I-E型CRISPR-Cas系统的序列完整性，根据基因鉴定结果，收集了两株包含完整I-E型CRISPR-Cas系统的链霉菌菌株；然后，证明了阿维链霉菌中的CRISPR-Cas系统在大肠杆菌中不具有活性，且阿维链霉菌中CRISPR-Cas系统无法编辑自身基因组；此外，鉴定了阿维链霉菌噬菌体phiSASD1的两个毒力蛋白holin和endolysin对阿维链霉菌、大肠杆菌的生长有极强的抑制作用，有被开发为抑菌剂的可能。