Collonnier Cécile, Guyon-Debast Anouchka, Maclot François, Mara Kostlend, Charlot Florence, Nogué Fabien
INRA Centre de Versailles-Grignon, IJPB (UMR1318) - route de St-Cyr, 78026 Versailles cedex, France.
INRA Centre de Versailles-Grignon, IJPB (UMR1318) - route de St-Cyr, 78026 Versailles cedex, France.
Methods. 2017 May 15;121-122:103-117. doi: 10.1016/j.ymeth.2017.04.024. Epub 2017 May 4.
Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.
除了在人类和动物治疗中发挥主要作用外，CRISPR-Cas9系统也已成为植物研究和植物育种的重要工具。农艺学应用依赖于对基因失活和基因修饰的掌握。然而，虽然通过CRISPR-Cas9系统诱导的靶向双链断裂（DSB）的非同源末端连接（NHEJ）介导的修复来敲除基因已经相当成熟，但在高等植物中通过同源驱动修复或末端连接进行基因敲入仍然难以高效实现。在本综述中，我们描述了可以测试的不同方法，以提高CRISPR诱导的植物基因修饰效率，包括使用最佳转化和再生方案、设计合适的引导RNA和供体模板以及选择核酸酶和递送方式。我们还介绍了在靶细胞中引导DNA修复途径可以采取的措施，并展示了如何将小立碗藓用作模式植物，以更好地理解涉及哪些DNA修复机制，以及这些知识最终如何用于定义更高效的CRISPR诱导基因敲入策略。
