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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.

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