Kirchner Thomas W, Niehaus Markus, Debener Thomas, Schenk Manfred K, Herde Marco
Institute of Plant Nutrition, Leibniz Universitaet Hannover, Hannover, Germany.
Institute for Plant Genetics, Leibniz Universitaet Hannover, Hannover, Germany.
PLoS One. 2017 Sep 22;12(9):e0185429. doi: 10.1371/journal.pone.0185429. eCollection 2017.
A protocol for the induction of site-directed deletions and insertions in the genome of Brassica carinata with CRISPR is described. The construct containing the Cas9 nuclease and the guide RNA (gRNA) was delivered by the hairy root transformation technique, and a successful transformation was monitored by GFP fluorescence. PAGE analysis of an amplified region, presumably containing the deletions and insertions, demonstrated up to seven different indels in one transgenic root and in all analyzed roots a wildtype allele of the modified gene was not detectable. Interestingly, many of these mutations consisted of relatively large indels with up to 112 bp. The exact size of the deletions was determined to allow an estimation whether the targeted gene was not functional due to a considerable deletion or a frame shift within the open reading frame. This allowed a direct phenotypic assessment of the previously characterized roots and, in fact, deletions in FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1)-a gene with an expression pattern consistent with a role in root hair architecture-resulted in shorter root hairs compared to control roots ectopically expressing an allele of the gene that cannot be targeted by the gRNA in parallel to the CRISPR construct. As an additional line of evidence, we monitored BcFLA1 expression with qPCR and detected a significant reduction of the transcript in roots with an active CRISPR construct compared to the control, although residual amounts of the transcript were detected, possibly due to inefficient nonsense-mediated mRNA decay. Additionally, the presence of deletions and insertions were verified by Sanger sequencing of the respective amplicons. In summary we demonstrate the successful application of CRISPR/Cas9 in hairy roots of B. carinata, the proof of its effectiveness and its effect on the root hair phenotype. This study paves the way for experimental strategies involving the phenotypic assessment of gene lesions by CRISPR which do not require germline transmission.
本文描述了一种利用CRISPR技术在埃塞俄比亚芥基因组中诱导位点定向缺失和插入的方案。通过发根转化技术导入含有Cas9核酸酶和引导RNA（gRNA）的构建体，并通过绿色荧光蛋白（GFP）荧光监测成功转化情况。对一个推测含有缺失和插入的扩增区域进行聚丙烯酰胺凝胶电泳（PAGE）分析，结果显示在一个转基因根中最多有7种不同的插入缺失，并且在所有分析的根中均未检测到修饰基因的野生型等位基因。有趣的是，这些突变中有许多是相对较大的插入缺失，长度可达112 bp。确定缺失的确切大小，以便估计靶向基因是否由于相当大的缺失或开放阅读框内的移码而失去功能。这使得能够直接对先前表征的根进行表型评估，事实上，与平行于CRISPR构建体异位表达不能被gRNA靶向的该基因等位基因的对照根相比，类成束蛋白阿拉伯半乳聚糖蛋白1（BcFLA1）基因（其表达模式与在根毛结构中的作用一致）中的缺失导致根毛更短。作为额外的证据，我们通过定量聚合酶链反应（qPCR）监测BcFLA1的表达，发现与对照相比，具有活性CRISPR构建体的根中该转录本显著减少，尽管检测到了转录本的残留量，这可能是由于无义介导的mRNA降解效率低下所致。此外，通过对相应扩增子进行桑格测序验证了插入缺失的存在。总之，我们证明了CRISPR/Cas9在埃塞俄比亚芥发根中的成功应用、其有效性的证据及其对根毛表型的影响。本研究为通过CRISPR进行基因损伤表型评估的实验策略铺平了道路，这些策略不需要种系传递。
