Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen, Nakaku, Sakai, Osaka, 599-8531, Japan.
Plant Cell Rep. 2019 Apr;38(4):463-473. doi: 10.1007/s00299-018-2320-7. Epub 2018 Jul 13.
Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site. In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. 'SR-1') was achieved using binary vectors comprising two (T1-T2) or three (T1-T2-T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRB, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1-T2-derived lines and two T1-T2-T3-derived lines. Of these mutations, four T1-T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5' homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1-T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (< 153 bp) region around the gRNA target site. Even though some T1-T2-T3-derived lines introduced three different T-DNAs and modified the An2 gRNA target site, no signs of HR in the endogenous An2 could be observed.
双元载体中转座子的共转化实现了烟草中 CRISPR/Cas9 介导的同源重组（HR）。HR 发生在 gRNA 靶位点周围的有限区域。在这项研究中，使用包含两个（T1-T2）或三个（T1-T2-T3）独立 T-DNA 区域的二元载体实现了烟草（Nicotiana tabacum L. 'SR-1'）中 CRISPR/Cas9 介导的同源重组（HR）。用于 HR 供体的烟草乙酰乳酸合酶基因 SuRB，T-DNA1 包含 ΔSuRB，其缺失 SuRB 的 N 末端区域，并且由三个核苷酸取代（ATG 到 GCT；W568L）引起，导致除草剂氯磺隆（Cs）抗性，由潮霉素（Hm）抗性基因侧翼。T-DNA2 由 hSpCas9 基因和两个靶向 SuRB 和 An2 的 gRNA 插入物组成。对于第二个 HR 供体，烟草 An2 基因编码参与花青素生物合成的 MYB 转录因子，T-DNA3 具有 35S 启动子驱动的 An2 基因，缺失 3 号外显子，导致成功 HR 后花青素积累。在从 7462 个农杆菌接种的外植体中选择 Hm 和 Cs 抗性后，获得了 77 个独立系。其中，在 8 个 T1-T2 衍生系和 2 个 T1-T2-T3 衍生系中检测到内源 SuRB 的 ATG 到 GCT 取代。这些突变中，有四个 T1-T2 衍生系为双等位基因。所有 HR 事件均发生在随机整合的 T-DNA1 的内源 SuRB 和 5'同源臂上。在一个 T1-T2 衍生系中也发现了 SuRB 旁系同源物 SuRA 的 HR。对其 SuRA 靶向区域的序列分析表明，HR 发生在 gRNA 靶位点周围的有限（<153bp）区域。尽管一些 T1-T2-T3 衍生系引入了三个不同的 T-DNAs 并修饰了 An2 gRNA 靶位点，但在内源性 An2 中观察不到 HR 的迹象。
