Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.
Base editing with high efficiency in allotetraploid oilseed rape by A3A-PBE system.
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DOI：
10.1111/pbi.13444
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发表时间：
2021-01
影响因子：
13.8
通讯作者：
Hu Q
中科院分区：
文献类型：
--
作者：
Cheng H;Hao M;Ding B;Mei D;Wang W;Wang H;Zhou R;Liu J;Li C;Hu Q
CRISPR/Cas碱基编辑是一种新兴技术，可以在靶位点将核苷酸转化为另一种类型。在这项研究中，在异源四倍体甘蓝型油菜中建立并开发了由人A3 A胞苷脱氨酶与Cas9切口酶和尿嘧啶糖基化酶抑制剂融合组成的A3 A-PBE系统。我们设计了三种sgRNA分别靶向ALS、RGA和IAA 7基因。对于所有三个靶基因，碱基编辑效率被证明超过20%。目标测序结果显示，编辑窗口范围为PAM序列的C1至C10。ALS的碱基编辑的植物赋予高除草剂抗性，而RGA或IAA 7的碱基编辑的植物表现出降低的植物高度。所有碱基编辑都可以从T0到T1代遗传。在所有三种sgRNA的靶位点处确认了几个插入缺失突变。此外，虽然在最潜在的脱靶位点没有检测到C到T的取代，但通过一些碱基编辑的植物和野生型植物之间的全基因组测序确定了大规模的SNP变异。这些结果表明，A3 A-PBE碱基编辑系统可以有效地将C替换转化为T替换，具有高编辑效率和扩大编辑窗口的优点。ALS、IAA 7和RGA基因的突变体可潜在地应用于赋予除草剂抗性以用于杂草控制或具有适合于机械收获的更好的植物结构。
CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T0 to T1 generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.
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影响因子：
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