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Genome editing (GE) technology has emerged as a multifaceted strategy that instantaneously popularised the mechanism to modify the genetic constitution of an organism. The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein-based genome editing (CRISPR/Cas) approach has huge potential for efficacious editing of genomes of numerous organisms. This framework has demonstrated to be more economical in contrast to mega-nucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) for its flexibility, versatility, and potency. The advent of sequence-specific nucleases (SSNs) allowed the precise induction of double-strand breaks (DSBs) into the genome, ensuring desired alterations through non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. Researchers have utilized CRISPR/Cas-mediated genome alterations across crop varieties to generate desirable characteristics for yield enhancement, enriched nutritional quality, and stressresistance. Here, we highlighted the recent progress in the area of nutritional improvement of crops via the CRISPR/Cas-based tools for fundamental plant research and crop genetic advancements. Application of this genome editing aids in unraveling the basic biology facts in plants supplemented by the incorporation of genome-wide association studies, artificial intelligence, and various bioinformatic frameworks, thereby providing futuristic model studies and their affirmations. Strategies for reducing the 'off-target' effects and the societal approval of genome-modified crops developed via this modern biotechnological approach have been reviewed.

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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.

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