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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 ( Cas 9-facilitated  H omologous  R ecombination  A ssembly)’ 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 ( M inimal  G enome 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.

CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA