Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.,Biotechnology Journal

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Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.
Biotechnology Journal ( IF 3.2 ) Pub Date : 2019-06-03 , DOI: 10.1002/biot.201900036
Xiang Jiao _{1,

2} , Yue Zhang _{1,

2} , Xiangjian Liu ₁ , Qi Zhang _{1,

2} , Sufang Zhang ₁ , Zongbao K Zhao ₁

Affiliation

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon-optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single-guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9-expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome-editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

中文翻译：

开发用于担子菌酵母红冬孢酵母基因组编辑的 CRISPR/Cas9 系统。

担子菌酵母圆红冬孢酵母（R. toruloides）已被探索作为生产脂质和类胡萝卜素的有前途的宿主。然而，由于缺乏有效的遗传工具，对该酵母的合理操作仍然很困难。在此，描述了用于 R. toruloides 基因组编辑的成簇规则间隔短回文重复序列 (CRISPR)/CRISPR 相关核酸酶 (Cas9) 系统的开发。首先，通过将含有密码子优化的 Cas9 基因的盒整合到基因组中，产生充分产生金黄色葡萄球菌来源的 Cas9 蛋白的圆红酵母菌株。同时，鉴定了两个 U6 基因，预测了两个 U6 启动子，并确认了单引导 RNA (sgRNA) 与 U6b 启动子的更好转录。接下来，分别针对CRTI、CAR2和CLYBL基因设计sgRNA盒，转化到Cas9表达的菌株中，发现超过60%的转化体成功插入和缺失（indel）突变。此外，当sgRNA盒包含侧翼为CRTI基因的两个同源臂的供体DNA时，通过同源重组发生基因敲除。因此，CRISPR/Cas9系统现已成为R. toruloides中强大的基因组编辑工具，这将促进功能基因组研究和先进的细胞工厂开发。

更新日期：2019-06-03

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