CRISPR/Cas9-mediated targeted T-DNA integration in rice
CRISPR/Cas9-mediated targeted T-DNA integration in rice
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DOI：
10.1007/s11103-018-00819-1
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发表时间：
2019-03-01
影响因子：
5.1
通讯作者：
Wang, Kan
中科院分区：
文献类型：
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作者：
Lee, Keunsub;Eggenberger, Alan L.;Wang, Kan
关键信息农杆菌介导的T-DNA整合是一种随机的整合方式，与CRISPR/Cas9系统相结合，可以实现T-DNA在水稻基因组中的精确定向整合。由于T-DNA优先整合到双链DNA断裂中，我们采用了CRISPR/Cas9系统来证明可以在水稻基因组中实现靶向T-DNA整合。使用标准农杆菌双元载体，我们构建了包含使用SpCas 9的CRISPR/Cas9系统和靶向水稻含AP 2结构域的蛋白基因Os 01 g 04020的外显子的gRNA的T-DNA。T-DNA还携带红色荧光蛋白和潮霉素抗性（hptII）基因。载体的一个版本具有由OsAct 2启动子驱动的hptII表达。为了检测靶向T-DNA插入事件，我们构建了另一个T-DNA，其具有邻近T-DNA右边界的无启动子hpt II基因，使得T-DNA整合到与hpt II基因符合读框的靶向外显子序列中将允许hpt II表达。我们的结果表明，这些构建体可以产生靶向的T-DNA插入，频率范围在4和5.3%之间的转基因愈伤组织事件，除了产生高频率（50-80%）的靶向indel突变。测序分析显示，五分之四的测序T-DNA/gDNA连接在靶位点携带全长T-DNA的单拷贝。我们的研究结果表明，农杆菌介导的转化结合CRISPR/Cas9系统可以有效地产生靶向T-DNA插入。
Key messageCombining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.AbstractAgrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.