Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Nucleic Acids Res. 2022 Oct 28;50(19):e109. doi: 10.1093/nar/gkac676.
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.
基因组可以通过 CRISPR/Cas9（成簇规律间隔短回文重复序列/CRISPR 相关肽 9）诱导的 DNA 双链断裂来编辑。然而，这种方法在插入或删除哺乳动物细胞中的长片段时效率不高。在这里，我们描述了一种简单的基因组编辑方法，称为转录偶联 Cas9 介导的编辑（TEd），它在删除基因组片段、插入/替换大片段 DNA 和引入哺乳动物细胞系中的点突变方面比经典 Cas9 介导的编辑（CEd）具有更高的效率。我们还发现，DNA 模板上的转录对于同源定向修复的促进至关重要，并且将 TEd 供体的转录物固定在靶向位点上进一步提高了编辑效率。TEd 在插入和删除长 DNA 片段方面的高效率扩展了 CRISPR 编辑哺乳动物基因组的应用。
