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Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.

ABSTRACT: Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR-Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR-Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We also describe quantification of genome-editing activity and off-target effects of the engineered CRISPR-Cpf1 in human cell lines using both T7 endonuclease I (T7E1) assay and targeted deep sequencing. This protocol enables rapid construction and identification of engineered crRNAs and Cpf1 mRNAs to enhance genome-editing efficiency using the CRISPR-Cpf1 system, as well as assessment of target specificity within 2 months. This protocol may also be appropriate for fine-tuning other types of CRISPR systems.

SUBMITTER: Li B

PROVIDER: S-EPMC6383568 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.

Li Bin B Li Bin B Zeng Chunxi C Dong Yizhou Y

Nature protocols 20180405 5

Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR-Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR-Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We ...[more]

PMID: 29622802

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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

Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.

Publications

Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.

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