Dataset Information

Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems.

ABSTRACT: Streptomyces strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabilities for gene editing in multiple Streptomyces strains, it has failed to work in some newly discovered strains and some important industrial strains. Additionally, the protospacer adjacent motif (PAM) recognition scope of this system sometimes limits its applications for generating precise site mutations and insertions. Here, we developed three efficient CRISPR-FnCas12a systems for multiplex genome editing in several Streptomyces strains. Each system exhibited advantages for different applications. The CRISPR-FnCas12a1 system was efficiently applied in the industrial strain Streptomyces hygroscopicus, in which SpCas9 does not work well. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. The CRISPR-FnCas12a3 system addressed the limitation of TTN PAM recognition in Streptomyces strains with high GC contents. In summary, all the CRISPR-FnCas12a systems developed in this study are powerful tools for precise and multiplex genome editing in Streptomyces strains.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7338789 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Efficient Multiplex Genome Editing in <i>Streptomyces</i> via Engineered CRISPR-Cas12a Systems.

Zhang Jun J Zhang Dan D Zhu Jie J Liu Huayi H Liang Shufang S Luo Yunzi Y

Frontiers in bioengineering and biotechnology 20200630

<i>Streptomyces</i> strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabi ...[more]

PMID: 32695773

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Project description: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 Streptococcus pyogenes CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces, it has a number of limitations, including the toxicity of SpCas9 expression in some important industrial Streptomyces 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 Francisella novicida) for multiplex genome editing and transcriptional repression in Streptomyces 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 Streptomyces coelicolor When no templates for HDR are present, random-sized DNA deletions are achieved by FnCpf1-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 FnCpf1 and SpCas9 exhibit different suitability in tested industrial Streptomyces species and show that FnCpf1 can efficiently promote HDR-mediated gene deletion in the 5-oxomilbemycin-producing strain Streptomyces hygroscopicus SIPI-KF, in which SpCas9 does not work well. Collectively, FnCpf1 is a powerful and indispensable addition to the Streptomyces CRISPR toolbox.IMPORTANCE Rapid, efficient genetic engineering of Streptomyces strains is critical for genome mining of novel natural products (NPs) as well as strain improvement. Here, a novel and high-efficiency Streptomyces genome editing tool is established based on the FnCRISPR-Cpf1 system, which is an attractive and powerful alternative to the S. pyogenes CRISPR-Cas9 system due to its unique features. When combined with HDR or NHEJ, FnCpf1 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, FnCpf1-based genome editing proves to be a highly efficient tool for genetic modification of some important industrial Streptomyces strains (e.g., S. hygroscopicus SIPI-KF) that cannot utilize the SpCRISPR-Cas9 system. We expect the CRISPR-Cpf1-assisted genome editing tool to accelerate discovery and development of pharmaceutically active NPs in Streptomyces as well as other actinomycetes.

Dataset Information

Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems.

Publications

Efficient Multiplex Genome Editing in <i>Streptomyces</i> via Engineered CRISPR-Cas12a Systems.

Similar Datasets

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