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

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 (ER1–6) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER1–6 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 EL1–6 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.

Toward Food Security in 2050: Gene Pyramiding for Climate-Smart Rice

“…Editing LOX3 and acetolactate synthase (OsALS) genes with TALENs' application has succeeded in precisely improving herbicide resistance and seed storability in rice [215]. Similarly, CRISPR/Cas9 has been used to manipulate rice properties such as the yield component [216][217][218][219], herbicide resistance [220,221], explosion resistance [219,[222][223][224][225][226][227][228], TGMS line development [229][230][231], stomata development [232,233], and amylose content modification [234][235][236][237][238].…”

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus

“…Alternatively, two other strategies also exist that still offer precise GE even without DRTs and DSBs. The first is called base editing (BE), a powerful technique facilitating gene replacement through HDR, enabling the precise substitution of a single base with a designated single nucleotide polymorphism (SNP) ( Sony et al, 2023 ). This technique facilitates the precise alteration of plant genomes in a programmed manner.…”

Transformative Approaches for Sustainable Weed Management: The Power of Gene Drive and CRISPR-Cas9

“…With genome editing tools at our disposal, the subject has gained more momentum. Genome-editing to impart herbicide resistance in crops has been attempted in maize, watermelon (Citrullus lantus L.), and oilseed rape (Brassica napus L.) to impart resistance against ALS (acetolactate synthase), inhibiting herbicides by base editing the ALS gene; in soybean to impart resistance to AHAS (acetohydroxyacid synthase), inhibiting herbicides by editing AHAS gene; in rice to impart resistance to glyphosate, th eALS inhibitor, the ACCase (Acetyl CoA carboxylase) inhibitor, and triketone herbicides by targeting EPSPS, ALS, ACCase and HPPD genes, respectively [111][112][113][114][115][116][117].…”