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G3 (Bethesda). 2019 Feb 21. pii: g3.200976.2018. doi: 10.1534/g3.118.200976. [Epub ahead of print]

Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.

Author information

1
Kwansei Gakuin University akihayashi@kwansei.ac.jp.
2
Kwansei Gakuin University.

Abstract

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

KEYWORDS:

CRISPR/Cas9; fission yeast; knock-in; point mutation; short-homology arm

PMID:
30755408
DOI:
10.1534/g3.118.200976
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