CRISPR-LbCas12a-mediated modification of citrus
CRISPR-LbCas12a-mediated modification of citrus
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DOI：
10.1111/pbi.13109
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发表时间：
2019-10-01
影响因子：
13.8
通讯作者：
Wang, Nian
中科院分区：
文献类型：
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作者：
Jia, Hongge;Orbovic, Vladimir;Wang, Nian
最近，来自Prevoella和Francisella的CRISPR-Cas12a(Cpf1)被改造成用于修饰植物基因组。在本报告中，我们首次利用来源于乳螺科细菌ND2006的CRISPR-LbCas12a(LbCpf1)来编辑柑橘基因组。首先，通过Xcc促进的农业渗入，利用LbCas12a成功地对邓肯葡萄柚中的CsPDS基因进行了修饰。接下来，用35S或YO启动子驱动的LbCas12a编辑CsLOB1启动子(EBEPthA4-CsLOBP)中的PthA4效应器结合元件。由于LbCas12a(TTTV)的保护基序邻近的基序允许crRNA作用于这两种类型的CsLOBP的保守区，因此选择一个单一的crRNA来靶向I型和II型CsLOBPs的保守区。CsLOB1是溃烂病的易感基因，由柑橘黄单胞菌相应的致病因子PthA4通过与EBEPthA4-CsLOBP结合而诱导。共产生7株转化35S-LbCas12a的邓肯植株，编号为#D(35)S1~#D(35)S7，创建10株YOO-LbCas12a转化的邓肯植株，命名为#D(姚)1~#D(姚)10。在3株35S-LbCas12a转化的邓肯植株(#D(35)S1、#D(35)S4和#D(35)S7)中观察到LbCas12a定向的EBEPthA4-CsLOBP修饰。然而，在姚-LbCas12a转化的植株中没有观察到LbCas12a介导的Indels。值得注意的是，含有最高突变率的转基因品系#D(35)S4减轻了Xcc Delta pthA4：dCsLOB1.4的感染。最后，没有观察到潜在的脱靶现象。因此，CRISPR-LbCas12a可作为柑橘基因组编辑的有力工具。
Recently, CRISPR-Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4-CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEPthA4-CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D(35)s1 to #D(35)s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #D(yao)1 to #D(yao)10. LbCas12a-directed EBEPthA4-CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D(35)s1, #D(35)s4 and #D(35)s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D(35)s4, which contains the highest mutation rate, alleviates Xcc Delta pthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. Therefore, CRISPR-LbCas12a can readily be used as a powerful tool for citrus genome editing.