Data Sheet 14_dCas9-SPO11-1 locally stimulates meiotic recombination in rice.pdf

IntroductionMeiotic crossovers shuffle the genetic information transmitted by the gametes. However, the potential to recover all the combinations of the parental alleles remains limited in most organisms, including plants, by the occurrence of only few crossovers per chromosome and a prominent bias in their spatial distribution. Thus, novel methods for stimulating recombination frequencies and/or modifying their location are highly desired to accelerate plant breeding. MethodsHere, we investigate the use of a dCas9-SPO11-1 fusion and clusters of 11 gRNAs to alter meiotic recombination in two chromosomal regions of a rice hybrid (KalingaIII/Kitaake). To accurately genotype rare recombinants in regions of few kbp, we improved the digital PCR-based pollen-typing method in parallel. ResultsExpression of the dCas9-SPO11-1 fusion protein under the ubiquitous ZmUbi1 promoter was obtained in leaves/anthers/meiocytes and found to complement the sterility of the Osspo11-1 mutant line. We observed a 3.27-fold increase over wild-type (p<0.001) of recombinant pollens in a transgenic hybrid line (7a) targeting a chromosome 7 region. In the offspring plant 7a1, a significant 2.05-fold increase (p=0.048) was observed in the central interval (7.2 kb) of the Chr. 7 target region. This stimulation of meiotic recombination is consistent with the expression of the dCas9-SPO11-1 fusion and gRNAs as well as with the ChIP-revealed binding of dCas9-SPO11-1 to the targeted region. In contrast, no stimulation was observed in other transgenic lines deficient in the above pre-requisite features, expressing the dCas9-SPO11-1 fusion but no gRNAs or targeting a Chr.9 region. DiscussionThese results open new avenues to locally stimulate meiotic recombination in crop genomes and paves the way for a future implementation in plant breeding programs.引言 减数分裂交换（Meiotic crossovers）可使配子所传递的遗传信息发生重组。然而，在多数包括植物在内的生物中，每条染色体仅发生少量交换且其空间分布存在显著偏倚，导致获取亲本等位基因全部组合的潜力仍受限制。因此，亟需开发可提升重组频率或改变重组位点的新方法，以加速植物育种进程。 方法 本研究探究了dCas9-SPO11-1融合蛋白与11条向导RNA（gRNAs）簇在水稻杂交品种（KalingaIII/Kitaake）的两个染色体区域内调控减数分裂重组的效果。为精准对数千碱基对区域内的稀有重组体进行基因分型，我们同时优化了基于数字PCR的花粉分型方法。 结果 将dCas9-SPO11-1融合蛋白置于组成型表达的ZmUbi1启动子调控下，可在叶片、花药及减数分裂细胞中实现其表达，并能够互补Osspo11-1突变体株系的不育表型。针对靶向7号染色体区域的转基因杂交株系7a，其重组花粉的数量较野生型提升了3.27倍（p<0.001）。在其后代植株7a1中，7号染色体靶区域的中央间隔区（7.2 kb）内的重组频率显著提升2.05倍（p=0.048）。这种减数分裂重组的激活效果与dCas9-SPO11-1融合蛋白及向导RNA的表达情况，以及染色质免疫沉淀（ChIP）实验所证实的dCas9-SPO11-1与靶区域的结合结果一致。与之相反，其他不具备上述必要特征的转基因株系（仅表达dCas9-SPO11-1融合蛋白但未表达向导RNA，或靶向9号染色体区域）未观察到重组频率提升现象。 讨论 本研究结果为在作物基因组中靶向激活减数分裂重组提供了新的思路，并为未来将该技术应用于植物育种项目铺平了道路。