CRISPR/Cas9-mediated homology donor repair base editing confers glyphosate resistance to rice (Oryza sativa L.)

Sonia Khan Sony; Tanushri Kaul; Khaled Fathy Abdel Motelb; Arulprakash Thangaraj; Jyotsna Bharti; Rashmi Kaul; Rachana Verma; Mamta Nehra

doi:10.3389/fpls.2023.1122926

CRISPR/Cas9-mediated homology donor repair base editing confers glyphosate resistance to rice (Oryza sativa L.)

Front Plant Sci. 2023 Mar 7:14:1122926. doi: 10.3389/fpls.2023.1122926. eCollection 2023.

Authors

Sonia Khan Sony¹, Tanushri Kaul¹, Khaled Fathy Abdel Motelb¹, Arulprakash Thangaraj¹, Jyotsna Bharti¹, Rashmi Kaul¹, Rachana Verma¹, Mamta Nehra¹

Affiliation

¹ Nutritional Improvement of Crops Group, Plant Molecular Biology Division, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.

Abstract

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 via 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-mOsEPSPS) and modification of phosphoenolpyruvate-binding site in the native OsEPSPS 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 OsEPSPS-NICTK-1_pCRISPR-Cas9 construct harboring rice codon-optimized SpCas9 along with OsEPSPS-HDR template was transformed into rice. Stable homozygous T₂ edited rice lines revealed significantly high degree of glyphosate-resistance both in vitro (4 mM/L) and field conditions (6 ml/L; Roundup Ready) in contrast to wild type (WT). Edited T₂ rice lines (ER_1-6) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER_1-6 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_1-6 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 OsEPSPS 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.

Keywords: CRISPR-Cas9; EPSP synthase; glyphosate; shikimate; weed; yield.

Grants and funding

The work was supported by Indian Council of Agricultural Research (ICAR) - National Agricultural Science Fund (NASF) (grant number NASF/GTR-7025/2018-19) and ICGEB for the provision of extramural and core funds, respectively, to perform this piece of work.