Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation.
Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation.
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第一代杨树中 CRISPR/Cas9 介导的高效定点诱变
DOI：
10.1038/srep12217
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发表时间：
2015-07-20
影响因子：
4.6
通讯作者：
Luo K
中科院分区：
文献类型：
--
作者：
Fan D;Liu T;Li C;Jiao B;Li S;Hou Y;Luo K
最近，使用II型簇状规则间隔短回文重复序列(CRISPR)相关蛋白(Cas)系统的RNA引导的基因组编辑已被应用于几个草本植物物种的基因组编辑。然而，目前还不清楚该系统是否可以用于木本植物的基因组编辑。本研究利用CRISPR/Cas9系统对木本植物毛白杨的基因组编辑和定向基因突变进行了研究。设计了4个引导RNAs(GRNAs)，分别针对杨树植烯脱饱和酶基因8(PtoPDS)的不同的基因组位置，然后是Protspacer相邻基序(PAM)。农杆菌介导法转化杨树后，观察到明显的白化表型。通过分析RNA引导的基因组编辑事件，59个PCR克隆中有30个是纯合子突变，2个是杂合突变，这些靶点的突变效率估计为51.7%。我们的数据表明，Cas9/sgRNA系统可以被用来精确编辑基因组序列，并有效地在木本植物中产生敲除突变。
Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species,Populus tomentosaCarr.viathe CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). AfterAgrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.