title: CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.
abstract: 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.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China University of the Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jianting
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China University of the Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Ronghai
authors.initials: R
authors.lastname: Wu
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China xlxue@sibs.ac.cn.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Xue
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China qin@sibs.ac.cn.
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Qin
mesh_terms: D020029:Base Pairing / Q000235:genetics*; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004270:DNA, Circular; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D059765:Homologous Recombination / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D064130:CRISPR-Associated Proteins; D004269:DNA, Bacterial; D004270:DNA, Circular