Rodríguez-López, M;
Cotobal, C;
Fernández-Sánchez, O;
Borbarán Bravo, N;
Oktriani, R;
Abendroth, H;
Uka, D;
... Bähler, J; + view all
(2017)
A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 3; peer review: 2 approved].
Wellcome Open Research
, 1
, Article 19. 10.12688/wellcomeopenres.10038.3.
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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.
| Type: | Article |
|---|---|
| Title: | A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast [version 3; peer review: 2 approved] |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.12688/wellcomeopenres.10038.3 |
| Publisher version: | http://dx.doi.org/10.12688/wellcomeopenres.10038.3 |
| Language: | English |
| Additional information: | © 2017 Rodríguez-López M et al. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. An earlier version is available at http://discovery.ucl.ac.uk/10037808/ |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Genetics, Evolution and Environment |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1539816 |
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