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.

中文翻译：

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转录偶联的供体 DNA 表达增加同源重组以进行有效的基因组编辑

基因组可以通过 CRISPR/Cas9 [成簇的规则间隔短回文重复序列 (CRISPR)/CRISPR 相关肽 9] 诱导的 DNA 双链断裂刺激的同源重组进行编辑。然而，这种方法对于在哺乳动物细胞中插入或删除长片段是低效的。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)，在删除基因组片段、插入/替换大 DNA 片段和引入方面可以实现比经典 Cas9 介导的编辑 (CEd) 更高的效率哺乳动物细胞系的点突变。我们还发现 DNA 模板上的转录对于促进同源定向修复至关重要，并且将来自 TEd 供体的转录物连接到目标位点进一步提高了编辑效率。