Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing
Kaixuan Gao, Xuedi Zhang, Zhenwu Zhang, Xiangyu Wu, Yan Guo, Pengchong Fu, Angyang Sun, Ju Peng, Jie Zheng, Pengfei Yu, Tengfei Wang, Qinying Ye, Jingwei Jiang, Haopeng Wang, Chao‐Po Lin, Guanjun Gao
Abstract
Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.