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Publication Detail
A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 3; peer review: 2 approved]
  • Publication Type:
    Journal article
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  • Authors:
    Rodríguez-López M, Cotobal C, Fernández-Sánchez O, Borbarán Bravo N, Oktriani R, Abendroth H, Uka D, Hoti M, Wang J, Zaratiegui M, Bähler J
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  • Journal:
    Wellcome Open Research
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  • Addresses:
    Research Department of Genetics, Evolution and Environment, University College London, London, UK.
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.
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