Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions
Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions
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DOI：
10.1016/j.ymben.2020.07.008
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发表时间：
2020-11-01
影响因子：
8.4
通讯作者：
Stephanopoulos, G.
中科院分区：
文献类型：
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作者：
Abdel-Mawgoud, A. M.;Stephanopoulos, G.
解脂耶氏酵母是一种重要的产油酵母，在油脂生物技术中具有广阔的应用前景。利用Y的全部潜力。然而，解脂酶受限于其对遗传操作的固有不相容性。与酿酒酵母（Saccharomycescerevisiae）相比，Y.解脂酵母在同源介导的DNA修复方面较差，因此在同源重组方面较差，这限制了该酵母中的位点特异性基因编辑。最近开发的基于CRISPR/Cas9的方法使用tRNA-sgRNA融合成功地编辑了Y中的一些基因组位点。解脂尽管如此，大多数其他测试的基因座或者编辑失败，或者使用这些方法实现了编辑但效率非常低。使用二级RNA结构预测的工具，我们能够改进用于在此类方法中表达单向导RNA（sgRNA）的tRNA-sgRNA融合物的设计。这导致在基因编辑失败或使用以前的方法以非常低的效率编辑的染色体基因座上进行高效的CRISPR/cas9基因编辑。此外，我们表征了我们新设计的tRNA-sgRNA融合体对于染色体基因整合和缺失的基因编辑性能。因此，这项研究提出了一种有效的CRISPR/Cas9介导的基因编辑工具，用于解脂耶氏酵母的高效基因工程。
Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica.