Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens
Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens
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DOI：
10.1007/s00294-021-01201-3
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发表时间：
2021-07
期刊：
影响因子：
2.5
通讯作者：
Maierdan Palihati;Hideo Tsubouchi;Bilge Argunhan;Rei Kajitani;Omirgul Bakenova;Yong-Woon Han;Yasuto Murayama;T. Itoh;H. Iwasaki
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文献类型：
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作者：
Maierdan Palihati;Hideo Tsubouchi;Bilge Argunhan;Rei Kajitani;Omirgul Bakenova;Yong-Woon Han;Yasuto Murayama;T. Itoh;H. Iwasaki
在这里，我们报告了能够对担子菌门酵母 Naganishia liquifaciens 进行基因改造的方法的开发。基因打靶方法采用电穿孔，PCR 产物两侧是与靶标同源的 80 bp 序列。该方法与新设计的 CRISPR-Cas9 系统相结合，通常可以实现 80% 的基因打靶效率。我们进一步探索了这种同源重组（HR）介导的基因靶向的遗传要求。 Ku70（DNA 双链断裂修复非同源末端连接 (NHEJ) 途径的主要组成部分）的缺失几乎完全消除了标记的不准确整合。具有短同源性 (80 bp) 的基因靶向几乎完全依赖于 Rad52，Rad52 是子囊菌、酿酒酵母和粟酒裂殖酵母中 HR 的重要组成部分。相比之下，RecA 同源物 Rad51 在 HR 中进行同源搜索和链交换，无论同源长度（80 bp 或 1 kb）如何，在基因打靶中发挥相对较小的作用。然而，Rad51 和 Rad52 的缺失完全消除了基因靶向。与子囊菌门酵母不同，N 中不存在 Rad52。液化菌对电离辐射仅具有轻微的敏感性。这些与 Rad52 缺失相关的特征让人想起在小鼠身上的发现。
Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast,Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 inN. liquefaciensconferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.