A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast
| Autor: | Jürg Bähler, María Rodríguez-López, Mimoza Hoti, R. Oktriani, Jin Wang, Natalia Borbarán Bravo, Oscar Fernández-Sánchez, Cristina Cotobal, Dardan Uka, Mikel Zaratiegui, Heike Abendroth |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Bioinformatics Medicine (miscellaneous) PCR primer design Biology General Biochemistry Genetics and Molecular Biology chemistry.chemical_compound 03 medical and health sciences Plasmid 0302 clinical medicine Genome editing genome editing CRISPR Selectable marker Genetics gene deletion Cas9 Articles Method Article biology.organism_classification Restriction enzyme 030104 developmental biology chemistry Schizosaccharomyces pombe Nourseothricin sgRNA CRISPR-Cas9 mutagenesis 030217 neurology & neurosurgery S. pombe |
| Zdroj: | Wellcome Open Research |
| ISSN: | 2398-502X |
| DOI: | 10.12688/wellcomeopenres.10038.2 |
| 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: | OpenAIRE |
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