The Pangenomics Lab, School of Science, RMIT University, Melbourne 3000, Australia.
Int J Mol Sci. 2021 Jan 1;22(1):396. doi: 10.3390/ijms22010396.
The current genome editing system Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9) has already confirmed its proficiency, adaptability, and simplicity in several plant-based applications. Together with the availability of a vast amount of genome data and transcriptome data, CRISPR/Cas9 presents a massive opportunity for plant breeders and researchers. The successful delivery of ribonucleoproteins (RNPs), which are composed of Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) and are used in combination with various transformation methods or lately available novel nanoparticle-based delivery approaches, allows targeted mutagenesis in plants species. Even though this editing technique is limitless, it has still not been employed in many plant species to date. Chickpea is the second most crucial winter grain crop cultivated worldwide; there are currently no reports on CRISPR/Cas9 gene editing in chickpea. Here, we selected the () and () genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The represents a key enzyme involved in phenylpropanoid metabolism in the lignin biosynthesis pathway. It regulates the accumulation of lignin under stress conditions in several plants. The is a MYB transcription factor which is part of regulating circadian rhythm in plants. The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted mutagenesis in chickpea. Results showed high-efficiency editing was achieved for gene in vivo compared to the gene. This study will help unravel the role of these genes under drought stress and understand the complex drought stress mechanism pathways. This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene editing of drought tolerance associated genes.
当前的基因组编辑系统簇状规律间隔短回文重复 CRISPR/Cas9（CRISPR/Cas9）已经在几种基于植物的应用中证实了其高效性、适应性和简单性。再加上大量基因组数据和转录组数据的可用性，CRISPR/Cas9 为植物育种者和研究人员提供了巨大的机会。核糖核蛋白（RNP）的成功传递，其由 Cas9 酶和合成设计的单指导 RNA（sgRNA）组成，并与各种转化方法或最近可用的新型基于纳米粒子的传递方法结合使用，可在植物物种中实现靶向突变。尽管这种编辑技术是无限的，但迄今为止，它在许多植物物种中仍未得到应用。鹰嘴豆是全球第二大冬季粮食作物；目前尚无关于鹰嘴豆 CRISPR/Cas9 基因编辑的报道。在这里，我们选择了 （）和 （）基因，这两个基因都与鹰嘴豆原生质体的耐旱性有关。 代表木质素生物合成途径中苯丙烷代谢的关键酶。它在几种植物的应激条件下调节木质素的积累。 是植物生物钟调节的 MYB 转录因子的一部分。使用无 DNA 的 CRISPR/Cas9 编辑在鹰嘴豆原生质体中敲除这些选定的基因代表了在鹰嘴豆中实现靶向突变的一种新方法。结果表明，与 基因相比， 基因在体内的编辑效率更高。这项研究将有助于揭示这些基因在干旱胁迫下的作用，并了解复杂的干旱胁迫机制途径。这是首例利用 CRISPR/Cas9 无 DNA 基因编辑对鹰嘴豆原生质体耐旱相关基因进行的研究。
