UCL  IRIS
Institutional Research Information Service
UCL Logo
Please report any queries concerning the funding data grouped in the sections named "Externally Awarded" or "Internally Disbursed" (shown on the profile page) to your Research Finance Administrator. Your can find your Research Finance Administrator at http://www.ucl.ac.uk/finance/research/post_award/post_award_contacts.php by entering your department
Please report any queries concerning the student data shown on the profile page to:

Email: portico-services@ucl.ac.uk

Help Desk: http://www.ucl.ac.uk/ras/portico/helpdesk
Publication Detail
A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    methods-article
  • 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
  • Publication date:
    01/2016
  • Pagination:
    19, ?
  • Journal:
    Wellcome open research
  • Volume:
    1
  • Medium:
    Electronic-eCollection
  • Print ISSN:
    2398-502X
  • Language:
    eng
  • Addresses:
    Research Department of Genetics, Evolution and Environment, University College London, London, UK.
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.
Publication data is maintained in RPS. Visit https://rps.ucl.ac.uk
 More search options
UCL Researchers
Author
Genetics, Evolution & Environment
Author
Genetics, Evolution & Environment
Author
Genetics, Evolution & Environment
University College London - Gower Street - London - WC1E 6BT Tel:+44 (0)20 7679 2000

© UCL 1999–2011

Search by