Wellcome Open Research (Jan 2017)

A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 2; referees: 2 approved]

  • María Rodríguez-López,
  • Cristina Cotobal,
  • Oscar Fernández-Sánchez,
  • Natalia Borbarán Bravo,
  • Risky Oktriani,
  • Heike Abendroth,
  • Dardan Uka,
  • Mimoza Hoti,
  • Jin Wang,
  • Mikel Zaratiegui,
  • Jürg Bähler

DOI
https://doi.org/10.12688/wellcomeopenres.10038.2
Journal volume & issue
Vol. 1

Abstract

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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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