Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors
Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors
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DOI：
10.1007/s42994-020-00018-x
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发表时间：
2020-04-01
期刊：
影响因子：
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通讯作者：
Yang, Yinong
中科院分区：
文献类型：
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作者：
Molla, Kutubuddin A.;Shih, Justin;Yang, Yinong
CRISPR/Cas9介导的碱基编辑技术可以有效地在基因组中产生点突变，而不需要引入双链断裂(DSB)或提供用于同源定向修复(HDR)的DNA供体模板。在这项研究中，用腺嘌呤碱基编辑程序(ABES)在水稻原生质体和再生植株中快速产生两个不同基因OsWSL5和OsZEBRA3(Z3)的精确点突变。精确设计的点突变被稳定地遗传给下一代。这些单核苷酸改变导致了单一氨基酸的改变和相关的wsl5和z3表型，分别表现为白色条纹叶片和淡绿色/暗绿色叶片模式。通过自交和遗传分离，在短时间内获得了无转基因、碱基编辑的wsl5和z3突变体。我们注意到Z3基因座的一个新突变(V540A)也可以模拟Z3突变(S542P)的表型。此外，我们在ABE编辑窗口中观察到一些编辑的植物中出现了意想不到的非A/G或T/C突变。本研究的ABE载体和方法可以在一次转化中同时产生多个靶基因的点突变，为作物改良和植物生物学基础研究提供了一个有用的碱基编辑工具。
The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break (DSB) or supplying a DNA donor template for homology-directed repair (HDR). In this study, adenine base editors (ABEs) were used for rapid generation of precise point mutations in two distinct genes, OsWSL5, and OsZEBRA3 (Z3), in both rice protoplasts and regenerated plants. The precisely engineered point mutations were stably inherited to subsequent generations. These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern, respectively. Through selfing and genetic segregation, transgene-free, base edited wsl5 and z3 mutants were obtained in a short period of time. We noticed a novel mutation (V540A) in Z3 locus could also mimic the phenotype of Z3 mutation (S542P). Furthermore, we observed unexpected non- A/G or T/C mutations in the ABE editing window in a few of the edited plants. The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.