中文摘要：

编辑技术 CRISPR 的指导 RNA 的 DNA 定期聚类 interspaced 短 palindromic repeats/Cas9 被用来介绍双 stranded 闯入染色体并且在细菌指导随后的地点特定的插入 / 删除或基因材料的代替例如 Escherichia coli，链球菌肺病，和乳杆菌 reuteri。在这研究，我们建立了在 Streptomyces 基因操作为使用编辑 plasmid pKCcas9dO 的一个高效率的 CRISPR/Cas9 染色体，它包括目标特定的指南 RNA，优化 codon 的 cas9，和二个指导相同的修理模板。由交付编辑 plasmids 进模型紧张 Streptomyces coelicolor M145 的 pKCcas9dO 系列，通过一步舞 intergeneric 转移，我们完成了与60%100%的高效率在不同层次编辑的染色体，包括单个基因删除，例如 actII-orf4 ，整顿，和 glnR ，并且单个大尺寸的基因簇删除，扮演 21.3 kb ， undecylprodigiosin 红 31.6 kb ，和 Ca ²⁺-dependent 抗菌素例如 actinorhodin 的抗菌素 biosynthetic 簇 82.8 kb 。而且，我们也分别地与 54% 和 45% 的高效率认识到 actII-orf4 和整顿，并且 ACT 和红 biosynthetic 基因簇的同时的删除。最后，我们使用了这个系统介绍核苷酸点变化进 rpsL 基因，它与抵抗授与异种到链球菌。用这个系统，显著地，为一个轮染色体修正要求的时间被三分之一个减少或为常规方法的那些的一个一半。这些结果清楚地显示实质地编辑系统的确定的 CRISPR/Cas9 染色体改进与当前存在的方法相比编辑效率在的染色体 Streptomyces，和它在另外的放线菌种类为申请有诺言到染色体修正。

英文摘要：

The RNA-guided DNA editing technology CRISPRs （clustered regularly interspaced short palindrom- ic repeats）/Cas9 had been used to introduce double-stranded breaks into genomes and to direct sub- sequent site-specific insertions/deletions or the replacement of genetic material in bacteria, such as Escherichia coli, Streptococcus pneumonia, and Lactobacillus reuteri. In this study, we established a high-efficiency CRISPR/Cas9 genome editing plasmid pKCcas9dO for use in Streptomyces genetic manipulation, which comprises a target-specific guide RNA, a codon-optimized cas9, and two hom- ology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the gen- ome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actll-orf4, redD, and glnR, and single large-size gene cluster deletion, such as the antibiotic biosynthetic clusters of actinorhodin （ACT） （21.3 kb）, undecylprodigiosin （RED） （31.6 kb）, and Ca2＋- dependent antibiotic （82.8 kb）. Furthermore, we also realized simultaneous deletions of actll-orf4 and redD, and of the ACT and RED biosynthetic gene clusters with high efficiencies of 54% and 45%, respectively. Finally, we applied this system to introduce nucleotide point mutations into the rpsL gene, which conferred the mutants with resistance to streptomycin. Notably, using this system, the time required for one round of genome modification is reduced by one-third or one-half of those for conventional methods. These results clearly indicate that the established CRISPR/Cas9 genome editing system substantially improves the genome editing efficiency compared with the currently existing methods in Streptomyces, and it has promise for application to genome modification in other Actinomyces species.