Department of Biology, University of Florida, Gainesville, Florida, USA.
Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), Versailles, 78000, France.
Plant J. 2024 Sep;119(6):2753-2764. doi: 10.1111/tpj.16946. Epub 2024 Aug 18.
The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.
CRISPR 技术的发展为理解重要生物过程的进化和功能提供了强大的工具。在这里，我们展示了在雌雄异株的苔藓物种 Ceratodon purpureus 中成功的 CRISPR-Cas9 基因组编辑。我们利用来自远缘雌雄同体苔藓 Physcomitrium patens 的现有选择系统，通过在天然 U6 snRNA 启动子的表达下进行 CRISPR 靶向诱变，生成 APT 报告基因的敲除体。接下来，我们利用天然同源定向修复（HDR）途径，结合 CRISPR-Cas9，在 Ceratodon purpureus 的一个新开发的着陆点中，在一个内源性 RPS5A 启动子的表达下敲入两个报告基因。我们的结果表明，在 P. patens 中开发的分子工具可以扩展到其他苔藓植物，跨越这个生态重要且发育多样化的群体。这些发现为旨在确定苔藓植物和其他陆地植物之间关键功能变异的遗传基础的精确和强大实验铺平了道路。
