CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repression in Streptomyces.

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 by Cpf1-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 and Cas9 exhibit different suitability in tested industrial species and show that Cpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain SIPI-KF, in which Cas9 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.