Elorriaga Estefania, Klocko Amy L, Ma Cathleen, Strauss Steven H
Department of Forest Ecosystems and Society, Oregon State University, Corvallis, OR, United States.
Department of Biology, University of Colorado Colorado Springs, Colorado Springs, CO, United States.
Front Plant Sci. 2018 May 7;9:594. doi: 10.3389/fpls.2018.00594. eCollection 2018.
In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus ). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, (), and the two poplar orthologs of the floral organ identity gene (). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3-24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both and , each individual sgRNA-gene combination had a unique mutation spectrum ( < 0.001). An -sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones ( > 0.05), however, a -sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones ( < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential "off-target" sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.
为了培育出能可靠地控制基因的转基因树木，我们使用CRISPR/Cas9系统改变了杨树（属）正常开花所需的三个基因。我们设计了合成导向RNA（sgRNA）来靶向花分生组织特征基因的杨树同源物以及花器官特征基因的两个杨树直系同源物。我们用sgRNA和Cas9转基因产生了557个转基因事件，用Cas9但不用sgRNA产生了49个事件，并通过对两个等位基因进行桑格测序来分析所有事件。在来自带有sgRNA的事件的684个扩增子中，474个在两个等位基因中都有突变（77.5%）。我们对法国国家农业研究院（INRA）克隆717 - 1B4中的71个事件和INRA克隆353 - 53中的22个事件的两个直系同源物进行了测序，发现分别有67个（94.4%）和21个（95.5%）是双位点敲除。部分由于靶区域存在单核苷酸多态性（SNP），发现一个靶向直系同源物的sgRNA自身完全无活性（0%），但与第二个sgRNA一起构建时在产生缺失方面显示出一定活性（10.3 - 24.5%）。在只有一个sgRNA的事件的突变等位基因中，小插入/缺失（indel）突变很普遍（范围从94.3%到99.1%），而在有两个活性sgRNA的等位基因中，大缺失很普遍（小indel的突变等位基因平均比例为22.6%，大indel为77.4%）。对于和，每个单独的sgRNA - 基因组合都有独特的突变谱（<0.001）。一个带有两个sgRNA的 - sgRNA构建体在两个杨树克隆中的突变谱相似（>0.05），然而，一个带有单个sgRNA的 - sgRNA构建体在相同的两个克隆中产生的突变谱有显著差异（<0.001）。49个空载体对照事件在两个等位基因中都没有突变，在研究的58个转基因事件中，310个潜在的“脱靶”序列也没有突变。CRISPR/Cas9是在杨树中产生功能缺失突变的非常强大且精确的系统，应该能有效地培育出可靠的不育树木，这可能会促进监管机构、市场或公众对基因工程技术的接受。
