Dataset Information

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.

ABSTRACT: In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic 'scars'. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.

SUBMITTER: Rodriguez-Lopez M

PROVIDER: S-EPMC5445975 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.

Rodríguez-López María M Cotobal Cristina C Fernández-Sánchez Oscar O Borbarán Bravo Natalia N Oktriani Risky R Abendroth Heike H Uka Dardan D Hoti Mimoza M Wang Jin J Zaratiegui Mikel M Bähler Jürg J

Wellcome open research 20160101

In the fission yeast <i>Schizosaccharomyces pombe</i> the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic 'scars'. The published method involves manual design of the single guide RNA (sgRNA), and digestio ...[more]

PMID: 28612052

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Project description:Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3?mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications.IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3?mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.

Dataset Information

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.

Publications

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.

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