Mechanisms of precise genome editing using oligonucleotide donors.
Mechanisms of precise genome editing using oligonucleotide donors.
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DOI：
10.1101/gr.214775.116
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发表时间：
2017-07
期刊：
影响因子：
7
通讯作者：
Hendrickson EA
中科院分区：
文献类型：
--
作者：
Kan Y;Ruis B;Takasugi T;Hendrickson EA
可编程大范围核酸酶的使用正在改变基因组编辑和功能基因组学。CRISPR/Cas9的开发使得靶向基因组损伤可以以前所未有的轻松方式引入体内。在同源供体的存在下，这些病变通过同源定向修复（HDR）途径促进高效精确的基因组编辑（PGE）。然而，导致PGE产物形成的HDR（亚）途径的身份和层次仍然难以捉摸。在这里，我们建立了一个绿色到蓝色荧光蛋白转化系统，以系统地表征寡脱氧核苷酸（ODN）介导的PGE使用Cas9及其切口酶变体在人类细胞中。我们证明，不像双链DNA（dsDNA）的捐助者与中央异源，ODNs产生短的转换道高斯样分布。有趣的是，使用ODN供体的单切口诱导的PGE产生的转化段根据ODN和切口的相对链性而偏向于主要是单向或双向的。此外，ODNs仅在双向转化途径中物理掺入基因组中，而不是在单向转化途径中。在双链基因组损伤的存在下，优先利用单向转化途径，即使敲入突变理论上可以通过两种途径转化。总的来说，我们的研究结果表明，ODN介导的PGE利用合成依赖性链退火和单链DNA掺入途径。这两种途径都产生具有类高斯分布的短转换域。虽然优先利用合成依赖性链退火，但我们的工作明确地建立了人细胞中单链DNA掺入途径的存在。这项工作扩展了HDR介导的基因转换的范例，并建立了人类细胞中PGE的指导方针。
The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussian-like distributions. Interestingly, single-nick–induced PGE using ODN donors produced conversion tracts biased either mostly uni- or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.