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

Abstract

<i>Streptomyces</i> 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 <i>Streptococcus pyogenes</i> CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in <i>Streptomyces</i>, it has a number of limitations, including the toxicity of <i>Sp</i>Cas9 expression in some important industrial <i>Streptomyces</i> 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 <i>Francisella novicida</i>) for multiplex genome editing and transcriptional repression in <i>Streptomyces</i> 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 <i>Streptomyces coelicolor</i> When no templates for HDR are present, random-sized DNA deletions are achieved by <i>Fn</i>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 <i>Fn</i>Cpf1 and <i>Sp</i>Cas9 exhibit different suitability in tested industrial <i>Streptomyces</i> species and show that <i>Fn</i>Cpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain <i>Streptomyces hygroscopicus</i> SIPI-KF, in which <i>Sp</i>Cas9 does not work well. Collectively, <i>Fn</i>Cpf1 is a powerful and indispensable addition to the <i>Streptomyces</i> CRISPR toolbox.<b>IMPORTANCE</b> Rapid, efficient genetic engineering of <i>Streptomyces</i> strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency <i>Streptomyces</i> genome editing tool is established based on the <i>Fn</i>CRISPR-Cpf1 system, which is an attractive and powerful alternative to the <i>S. pyogenes</i> CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, <i>Fn</i>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, <i>Fn</i>Cpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial <i>Streptomyces</i> strains (e.g., <i>S. hygroscopicus</i> SIPI-KF) that cannot utilize the <i>Sp</i>CRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in <i>Streptomyces</i> as well as other actinomycetes.