Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast
Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast
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DOI：
10.1534/g3.118.200976
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发表时间：
2019-04-01
影响因子：
2.6
通讯作者：
Tanaka, Katsunori
中科院分区：
文献类型：
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作者：
Hayashi, Aki;Tanaka, Katsunori
CRISPR/Cas9系统能够通过诱导特定染色体靶点的双链断裂(DSB)来编辑大量生物的基因组。我们改进了CRISPR/Cas9系统，通过将其与基于分裂酵母中非规范同源定向DNA修复(HDR)的基因组编辑相结合，简化了将点突变或标签序列直接引入染色体的过程。我们构建了方便的克隆载体，它带有引导RNA(GRNA)表达模块，或人源化化脓性链球菌Cas9基因，在可诱导启动子的控制下表达，以避免不必要的表达，或同时具有gRNA和Cas9基因。利用这个系统，我们尝试了短同源基因介导的基因组编辑，发现HDR途径在不需要长供体DNA的情况下，在目标座位提供了高频的基因组编辑。利用短链寡核苷酸，我们成功地在两个靶基因中引入了高频点突变。我们还利用具有较短同源性的供体DNA将表位和GFP标记序列精确整合到靶基因座中，从而获得了表达N端标记的融合蛋白的细胞。该系统可加快裂解酵母基因组编辑的速度，并可应用于其他生物。
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.