The RNA-guided DNA editing technology CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas9 had been used to introduce double-stranded breaks into genomes and to direct subsequent 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 homology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the genome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actII-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 Ca 2+ -dependent antibiotic (82.8 kb). Furthermore, we also realized simultaneous deletions of actII-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.

has a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in, it has a number of limitations, including the toxicity of Cas9 expression in some important industrial strains and the need for complex expression constructs when targeting multiple genomic loci. To address these problems, in this study, we developed a high-efficiency CRISPR-Cpf1 system (from ) for multiplex genome editing and transcriptional repression in Using an all-in-one editing plasmid with homology-directed repair (HDR), our CRISPR-Cpf1 system precisely deletes single or double genes at efficiencies of 75 to 95% in When no templates for HDR are present, random-sized DNA deletions are achieved byCpf1-induced double-strand break (DSB) repair by a reconstituted nonhomologous end joining (NHEJ) pathway. Furthermore, a DNase-deactivated Cpf1 (ddCpf1)-based integrative CRISPRi system is developed for robust, multiplex gene repression using a single customized crRNA array. Finally, we demonstrate that Cpf1 andCas9 exhibit different suitability in tested industrial species and show thatCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain SIPI-KF, in whichCas9 does not work well. Collectively, Cpf1 is a powerful and indispensable addition to the CRISPR toolbox. Rapid, efficient genetic engineering of strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency genome editing tool is established based on the CRISPR-Cpf1 system, which is an attractive and powerful alternative to the CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, Cpf1 enables the creation of gene(s) deletion with high efficiency. Furthermore, a ddCpf1-based integrative CRISPRi platform is established for simple, multiplex transcriptional repression. Of importance,Cpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial strains (e.g., SIPI-KF) that cannot utilize the CRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in as well as other actinomycetes.