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	Cashra (cas9-facilitated homologous recombination assembly) method of constructing megabase-sized dna
	Zhou, Jianting1,2; Wu, Ronghai1,2; Xue, Xiaoli1; Qin, Zhongjun1
刊名	Nucleic acids research
	2016-08-19
卷号	44期号:14页码:9
ISSN号	0305-1048
DOI	10.1093/nar/gkw475
通讯作者	Xue, xiaoli(xlxue@sibs.ac.cn) ; Qin, zhongjun(qin@sibs.ac.cn)
英文摘要	Current dna assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized dnas (e.g. synthetic genomes). we have developed a new method designated 'cashra (cas9-facilitated homologous recombination assembly)' that directly uses large circular dnas in a one-step in vivo assembly process. the large circular dnas are co-introduced into saccharomyces cerevisiae by protoplast fusion, and they are cleaved by rna-guided cas9 nuclease to release the linear dna segments for subsequent assembly by the endogenous homologous recombination system. the cashra method allows efficient assembly of multiple large dna segments in vivo; thus, this approach should be useful in the last stage of genome construction. as a proof of concept, we combined cashra with an upstream assembly method (gibson procedure of genome assembly) and successfully constructed a 1.03 mb mge-syn1.0 (minimal genome of escherichia coli) that contained 449 essential genes and 267 important growth genes. we expect that cashra will be widely used in megabase-sized genome constructions.
WOS关键词	TRANSFORMATION-ASSOCIATED RECOMBINATION ; SACCHAROMYCES-CEREVISIAE ; ESCHERICHIA-COLI ; ONE-STEP ; VIBRIO-CHOLERAE ; REDUCED-GENOME ; YEAST ; CRISPR ; GENE ; SYSTEMS
WOS研究方向	Biochemistry & Molecular Biology
WOS类目	Biochemistry & Molecular Biology
语种	英语
出版者	OXFORD UNIV PRESS
WOS记录号	WOS:000382999900004
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2375638
专题	中国科学院大学
通讯作者	Xue, Xiaoli; Qin, Zhongjun
作者单位	1.Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Plant Physiol & Ecol, Key Lab Synthet Biol, Shanghai 200032, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Zhou, Jianting,Wu, Ronghai,Xue, Xiaoli,et al. Cashra (cas9-facilitated homologous recombination assembly) method of constructing megabase-sized dna[J]. Nucleic acids research,2016,44(14):9.
APA	Zhou, Jianting,Wu, Ronghai,Xue, Xiaoli,&Qin, Zhongjun.(2016).Cashra (cas9-facilitated homologous recombination assembly) method of constructing megabase-sized dna.Nucleic acids research,44(14),9.
MLA	Zhou, Jianting,et al."Cashra (cas9-facilitated homologous recombination assembly) method of constructing megabase-sized dna".Nucleic acids research 44.14(2016):9.

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