First Report of CRISPR/Cas9 Mediated DNA-Free Editing of 4CL and RVE7 Genes in Chickpea Protoplasts.
First Report of CRISPR/Cas9 Mediated DNA-Free Editing of 4CL and RVE7 Genes in Chickpea Protoplasts.
复制标题
DOI：
10.3390/ijms22010396
复制
发表时间：
2021-01-01
影响因子：
5.6
通讯作者：
Mantri N
中科院分区：
文献类型：
--
作者：
Badhan S;Ball AS;Mantri N
目前的基因组编辑系统CRISPR/Cas9（Regularly Interspaced Short Palindromic Repeats Cas9）已经在几种基于植物的应用中证实了其熟练性，适应性和简单性。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。由Cas9酶和合成设计的单向导RNA（sgRNA）组成并与各种转化方法或最近可用的基于纳米颗粒的新型递送方法组合使用的核糖核蛋白（RNP）的成功递送允许在植物物种中进行靶向诱变。尽管这种编辑技术是无限的，但迄今为止，它仍然没有在许多植物物种中使用。鹰嘴豆是全球种植的第二大冬季粮食作物，目前还没有关于鹰嘴豆中CRISPR/Cas9基因编辑的报道。在这里，我们选择了4-香豆酸连接酶（4CL）和Reveille 7（RVE 7）基因，这两个基因都与鹰嘴豆原生质体中的CRISPR/Cas9编辑的耐旱性相关。4CL代表了木质素生物合成途径中参与苯丙素代谢的关键酶。它在几种植物的胁迫条件下调节木质素的积累。RVE 7是MYB转录因子，是调节植物昼夜节律的一部分。使用无DNA 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诱变的新方法。结果表明，与4CL基因相比，RVE 7基因在体内实现了高效编辑。该研究将有助于揭示这些基因在干旱胁迫下的作用，并有助于理解复杂的干旱胁迫机制。这是首次在鹰嘴豆原生质体中利用CRISPR/Cas9 DNA自由基因编辑耐旱相关基因的研究。
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 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes, both associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast. The 4CL 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 RVE7 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 RVE7 gene in vivo compared to the 4CL 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.
登录
查看更多内容
影响因子：
3.5
作者：
Cao Y;Han Y;Li D;Lin Y;Cai Y
通讯作者：
Cai Y
影响因子：
1.9
作者：
Cheng, Ninghui;Nakata, Paul A.
通讯作者：
Nakata, Paul A.
干旱胁迫下玉米叶片木质素生物合成候选基因的差异表达
影响因子：
2.4
作者：
Hu, Y.;Li, W-Ch.;Fu, F-L.
通讯作者：
Fu, F-L.
影响因子：
4.6
作者：
Johansen, Ida Elisabeth;Liu, Ying;Petersen, Bent Larsen
通讯作者：
Petersen, Bent Larsen
影响因子：
12.3
作者：
Carroll D;Charo RA
通讯作者：
Charo RA