Sony Sonia Khan, Kaul Tanushri, Motelb Khaled Fathy Abdel, Thangaraj Arulprakash, Bharti Jyotsna, Kaul Rashmi, Verma Rachana, Nehra Mamta
Nutritional Improvement of Crops Group, Plant Molecular Biology Division, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
Front Plant Sci. 2023 Mar 7;14:1122926. doi: 10.3389/fpls.2023.1122926. eCollection 2023.
Globally, CRISPR-Cas9-based genome editing has ushered in a novel era of crop advancements. Weeds pose serious a threat to rice crop productivity. Among the numerous herbicides, glyphosate [N-(phosphonomethyl)-glycine] has been employed as a post-emergent, broad-spectrum herbicide that represses the shikimate pathway inhibition of EPSPS (5'-enolpyruvylshikimate-3-phosphate synthase) enzyme in chloroplasts. Here, we describe the development of glyphosate-resistant rice lines by site-specific amino acid substitutions (G172A, T173I, and P177S: GATIPS-m) and modification of phosphoenolpyruvate-binding site in the native gene employing fragment knockout and knock-in of homology donor repair (HDR) template harboring desired mutations through CRISPR-Cas9-based genome editing. The indigenously designed two-sgRNA -NICTK-1_pCRISPR-Cas9 construct harboring rice codon-optimized Cas9 along with -HDR template was transformed into rice. Stable homozygous T edited rice lines revealed significantly high degree of glyphosate-resistance both (4 mM/L) and field conditions (6 ml/L; Roundup Ready) in contrast to wild type (WT). Edited T rice lines (ER) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER lines exhibited increased aromatic amino acid contents (Phe, two-fold; Trp, 2.5-fold; and Tyr, two-fold) than WT. Interestingly, glyphosate-resistant Cas9-free EL rice lines displayed a significant increment in grain yield (20%-22%) in comparison to WT. Together, results highlighted that the efficacy of GATIPS mutations in has tremendously contributed in glyphosate resistance (foliar spray of 6 ml/L), enhanced aromatic amino acids, and improved grain yields in rice. These results ensure a novel strategy for weed management without yield penalties, with a higher probability of commercial release.
在全球范围内，基于CRISPR-Cas9的基因组编辑开启了作物改良的新时代。杂草对水稻作物的生产力构成严重威胁。在众多除草剂中，草甘膦[N-(膦酰基甲基)甘氨酸]已被用作芽后广谱除草剂，它抑制叶绿体中莽草酸途径对5'-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）的抑制作用。在此，我们描述了通过位点特异性氨基酸替换（G172A、T173I和P177S：GATIPS-m）以及利用基于CRISPR-Cas9的基因组编辑对天然基因中磷酸烯醇丙酮酸结合位点进行片段敲除和携带所需突变的同源供体修复（HDR）模板的敲入来培育抗草甘膦水稻品系。将含有水稻密码子优化的Cas9以及-HDR模板的自主设计的双sgRNA -NICTK-1_pCRISPR-Cas9构建体转化到水稻中。稳定的纯合T编辑水稻品系在实验室条件下（4 mM/L）和田间条件下（6 ml/L；农达）均显示出比野生型（WT）显著更高的草甘膦抗性。与处理后的WT相比，具有增强草甘膦抗性的编辑T水稻品系（ER）中内源性莽草酸水平较低（低14.5倍），但与WT相当。ER品系的芳香族氨基酸含量（苯丙氨酸，增加两倍；色氨酸，增加2.5倍；酪氨酸，增加两倍）高于WT。有趣的是，与WT相比，不含Cas9的抗草甘膦EL水稻品系的籽粒产量显著增加（20%-22%）。总之，结果表明GATIPS突变在水稻中对草甘膦抗性（叶面喷施6 ml/L）、增强芳香族氨基酸以及提高籽粒产量有巨大贡献。这些结果确保了一种无产量损失的杂草管理新策略，具有更高的商业推广可能性。
