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CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae.

ABSTRACT: Cpf1 represents a novel single RNA-guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues - Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) - for genome editing of Saccharomyces cerevisiae. These Cpf1-based systems enable fast and reliable introduction of donor DNA on the genome using a two-plasmid-based editing approach together with linear donor DNA. LbCpf1 and FnCpf1 displayed editing efficiencies comparable with the CRISPR/Cas9 system, whereas AsCpf1 editing efficiency was lower. Further characterization showed that AsCpf1 and LbCpf1 displayed a preference for their cognate crRNA, while FnCpf1-mediated editing with similar efficiencies was observed using non-cognate crRNAs of AsCpf1 and LbCpf1. In addition, multiplex genome editing using a single LbCpf1 crRNA array is shown to be functional in yeast. This work demonstrates that Cpf1 broadens the genome editing toolbox available for Saccharomyces cerevisiae. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

SUBMITTER: Verwaal R

PROVIDER: S-EPMC5836994 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae.

Verwaal René R Buiting-Wiessenhaan Nathalie N Dalhuijsen Sacha S Roubos Johannes A JA

Yeast (Chichester, England) 20171112 2

Cpf1 represents a novel single RNA-guided CRISPR/Cas endonuclease system suitable for genome editing with distinct features compared with Cas9. We demonstrate the functionality of three Cpf1 orthologues - Acidaminococcus spp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1) and Francisella novicida U112 (FnCpf1) - for genome editing of Saccharomyces cerevisiae. These Cpf1-based systems enable fast and reliable introduction of donor DNA on the genome using a two-plasmid-based editing app ...[more]

PMID: 28886218

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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

CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae.

Publications

CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae.

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