Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.
Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.
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DOI：
10.1093/jxb/ery245
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发表时间：
2018-09-14
影响因子：
6.9
通讯作者：
Xia L
中科院分区：
文献类型：
--
作者：
Li S;Li J;Zhang J;Du W;Fu J;Sutar S;Zhao Y;Xia L
依赖于合成的链退火是Cpf1诱导的DSB在同源修复模板上修复的主要机制；这样的模板是可用的，并使水稻中有针对性的基因替换。最近发展起来的CRISPR/Cpf1系统扩大了基因组编辑的范围，正在成为植物功能基因组学和作物改良的一种替代有力的工具。Cpf1-CRISPR RNA(CrRNA)产生具有长的5‘-突起末端的双链DNA断裂(DSB)，这可能通过同源定向修复(HDR)促进修复模板的配对和插入，以进行靶向基因替换，并在特定的基因座上引入所需的DNA元件以用于作物改良。然而，由Cpf1-crRNA产生的DSB的HDR的潜在机制仍有待研究，而且HDR固有的低效率和外源供体DNA作为修复模板的可用性极大地阻碍了HDR在作物基因组精确编辑中的应用。在这里，我们提供了Cpf1诱导的DSB合成依赖修复的证据，这使我们能够准确地用预期的突变版本取代野生型ALS基因，该突变版本携带两个离散的点突变，从而使水稻对除草剂产生抗性。我们观察到，只有左侧同源臂的供体修复模板(DRT)足以进行精确的靶向等位基因替换，这有助于更好地理解植物HDR的机制，并极大地简化了DRT的设计，用于作物改良中的精确基因组编辑。
Synthesis-dependent strand annealing is a primary mechanism in the repair of Cpf1-induced DSBs on homology repair templates; such templates are available and enable targeted gene replacement in rice. The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.
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