CasHRAC: Cas9-facilitated homologous recombination for efficient assembly of circular DNAs up to sev

• 作 者: JiantingZhou;RonghaiWu;XiaoliXue;ZhongjunQin;
• 机 构: Key Laboratory of Synthetic Biology,Institute of Plant Physiology and Ecology,Shanghai Institutes fo
• 出 处: 中国生物工程学会第六次全国会员代表大会暨第九届学术年会
• 发表日期: 2015年期

　　Standardized DNA assembly have enabled a broad range of synthetic biology research.Still,the assembly of large constructs up to several hundred kilobases has been challenging,mainly due to the complex in vitro manipulation of long DNA segments.Here,we describe a new method designated “CasHRAC(Cas9-facilitated homologous recombination for assembly of circular DNAs)”,which allows direct usage of multiple large plasmids(> 100 kbp)for one-step DNA assembly in vivo.The large plasmids are gathered into the same yeast cell by protoplast fusion,and be cut by the RNA-guided Cas9 nuclease to release the linear DNA segments to join in assembly by taking advantage of the native homologous recombination system.As a proof-of-concept,we apply CasHRAC for assembly of plasmids with various length of insertions(10~298 kbp)into four DNA constructs,and the largest assembly is 660 kbp and contains all published Escherichia coli essential genes.This method complements and improves the current methods for large DNA assembly,especially in the whole genome assembly.