| Autor: |
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 |
| Jazyk: |
angličtina |
| Rok vydání: |
2017 |
| Předmět: |
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| Zdroj: |
Wellcome Open Research, Vol 1 (2017) |
| Druh dokumentu: |
article |
| ISSN: |
2398-502X |
| DOI: |
10.12688/wellcomeopenres.10038.3 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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