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

Rapid, efficient genetic engineering ofStreptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiencyStreptomyces genome editing tool is established based on theFn CRISPR-Cpf1 system, which is an attractive and powerful alternative to theS. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ,Fn 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,Fn Cpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrialStreptomyces strains (e.g.,S. hygroscopicus SIPI-KF) that cannot utilize theSp CRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs inStreptomyces as well as other actinomycetes.