One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces  

作　　者：He Huang Guosong Zheng Weihong Jiang Haifeng Hu Yinhua Lu 

机构地区：[1]Key Lab of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institute's for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China [2]Shanghai Institute of Pharmaceutical Industry,Shanghai 200040, China

出　　处：《Acta Biochimica et Biophysica Sinica》2015年第4期231-243,共13页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Basic Research Program of China （Nos. 2011CBA00800 and 2012CB721103）, National High Technology Research and Develop- ment Program of China （No. 2012AA022107）, National Natural Science Foundation of China （Nos. 31121001 and 31430004）, and Program of Chinese Academy of Sciences （No. KGZD-EW-606）.

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.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.

关 键 词：CRISPRs （clustered regularly interspaced short palindromic repeats） genome editing STREPTOMYCES 

分 类 号：Q78[生物学—分子生物学] G232[文化科学]