Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore MD 21205
Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore MD 21205.
Proc Natl Acad Sci U S A. 2017 Dec 12;114(50):E10745-E10754. doi: 10.1073/pnas.1711979114. Epub 2017 Nov 28.
The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
RNA 指导的 DNA 内切酶 Cas9 已成为基因组工程的强大工具。Cas9 在基因组中产生靶向双链断裂 (DSB)。通过含有所需编辑的合成供体 DNA 对 DSB 进行同源定向修复 (HDR)，可以实现特定突变的敲入 (精确基因组编辑)，但据报道 HDR 的效率存在差异。在这里，我们报告线性 DNA（单链和双链）参与一种高效的 HDR 机制，仅需与靶位点同源的约 35 个核苷酸即可引入从 1 到 1000 个核苷酸不等的编辑。我们通过使用 PCR 片段在人类细胞和小鼠胚胎中引入荧光蛋白标签来证明线性供体的实用性。我们发现修复是局部的、极性敏感的并且容易发生模板转换，这些特征与合成依赖性链退火的基因转换一致。我们的发现使我们能够为高效基因组编辑合理设计合成供体 DNA。
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