Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

doi:10.1002/biot.201900036

	Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides
	Jiao, Xiang 1,2; Zhang, Yue 1,2; Liu, Xiangjian 2; Zhang, Qi 1,2; Zhang, Sufang 2; Zhao, Zongbao K.2
刊名	BIOTECHNOLOGY JOURNAL
	2019-07-01
卷号	14 期号:7 页码:7
关键词	basidiomycetous yeasts CRISPR Cas9 genome editing Rhodosporidium toruloides transformation
ISSN号	1860-6768
DOI	10.1002/biot.201900036
通讯作者	Zhang, Sufang(zsfjxy@dicp.ac.cn) ; Zhao, Zongbao K.(zhaozb@dicp.ac.cn)
英文摘要	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.
资助项目	National Natural Science Foundation of China[31870042] ; National Natural Science Foundation of China[21721004] ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences[DMTO201701]
WOS关键词	OLEAGINOUS YEAST ; IN-VIVO ; GENES ; CAS9 ; TRANSFORMATION ; PROMOTERS
WOS研究方向	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
语种	英语
出版者	WILEY-V C H VERLAG GMBH
WOS记录号	WOS:000476825900015
资助机构	National Natural Science Foundation of China ; National Natural Science Foundation of China ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; National Natural Science Foundation of China ; National Natural Science Foundation of China ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; National Natural Science Foundation of China ; National Natural Science Foundation of China ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; National Natural Science Foundation of China ; National Natural Science Foundation of China ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences ; Dalian Institute of Chemical Physics, Chinese Academy of Sciences
内容类型	期刊论文
源URL	[http://cas-ir.dicp.ac.cn/handle/321008/173841]
专题	大连化学物理研究所_中国科学院大连化学物理研究所
通讯作者	Zhang, Sufang; Zhao, Zongbao K.
作者单位	1.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 2.Chinese Acad Sci, Dalian Inst Chem Phys, Div Biotechnol, Dalian 116023, Peoples R China
推荐引用方式 GB/T 7714	Jiao, Xiang,Zhang, Yue,Liu, Xiangjian,et al. Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides[J]. BIOTECHNOLOGY JOURNAL,2019,14(7):7.
APA	Jiao, Xiang,Zhang, Yue,Liu, Xiangjian,Zhang, Qi,Zhang, Sufang,&Zhao, Zongbao K..(2019).Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides.BIOTECHNOLOGY JOURNAL,14(7),7.
MLA	Jiao, Xiang,et al."Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides".BIOTECHNOLOGY JOURNAL 14.7(2019):7.