Mature, healthy, dry rice seeds (Oryza sativa L.) var. Samba Mahsuri were manually dehusked and disinfected with 70% (w/v) ethanol for 2 min, then treated with 2% sodium hypochlorite supplemented with Tween 20 (one drop) for 18 min with intermittent shaking, and then washed thrice to remove the sodium hypochlorite. Finally, seeds were dried and used as explants for callus preparation. Sterilized dried seeds (8–10 seeds per plate) were cultured on callus induction medium that comprised MS medium (Murashige and Skoog, 1962) supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.5 mg/L), Thidiazuron (TDZ) (0.1 mg/L), casein hydrolysate (0.3 g/L), dicamba (1.5 mg/L), and proline (1g/L) and incubated in the dark at a temperature of 25°C ± 2°C and related humidity (RH) of 50%–60%. After 4–5 days of incubation in the dark, small calli originating from the scutellar zone of endosperm were separated and incubated on fresh callus induction medium with similar medium compositions in the dark for an extended 15 days before being used for biolistic transformation (Kaul et al., 2021).

In silico assessment was performed to comprehend the impact of GATIPS (G172A, T17I, and P177S) mutation in the native OsEPSPS protein in imparting glyphosate resistance. Schrodinger suite: release 2017-2 (Schroüdinger, 2017) was utilized to carry out the computational analysis and in silico calculations. The protein sequence of OsEPSPS and two ligands (glyphosate and PEP) were selected for the computational analysis, and those molecules were retrieved from the protein data bank (https://www.rcsb.org). The OsEPSPS sequence was analyzed for conserved domain identification ( Figure 1A ), followed by retrieving its homologs in various monocot and dicot plant species from NCBI (NCBI Resource Coordinators, 2015). The multiple sequence alignment of different EPSPS sequences was performed to uncover the conserved regions using Bioedit (Hall, 2011). After alignment, the generated phylogenetic tree ( Figure 1B ) was inferred by employing the neighbor-joining/UPGMA: Unweighted pair group method with arithmetic mean method at 1,000 bootstrap iterations (Saitou and Nei, 1987). The spatial data files of the glyphosate and PEP structures were retrieved from the PDB (https://www.rcsb.org) for ligand preparations, and docking was performed using the Schrodinger suite. Cavity projections with ligand geometry and binding affinity of glyphosate and PEP with wild and mutant EPSPS proteins were also analyzed using Schrodinger suite ( Figures 1C–E ).

A robust marker-free pCAMBIA1300-based plant expression vector NICTK-1_pCRISPR-Cas9 (16.0 kb) was designed indigenously and synthesized by GeneArt: ThermoScientifc, USA). This vector harbored a transgene cassette of 6.6 kb comprising rice codon-optimized SpCas9 coding sequence (4.1 kb) flanked by nuclear localization signals (NLSs) at both ends. The Cas9 gene expression cassette was driven by a maize ubiquitin (pUbi) promoter (1.9 kb) along with nopaline synthase (NOS) (253-bp) terminator. The complete rice codon-optimized Cas9 expression cassette was cloned within the binary vector using Srf1-Srf1 restriction sites. Furthermore, two-sgRNA target sequences of OsEPSPS were selected in such a manner that they flanked the conserved PEP-binding active site ( Supplementary Figures S2 , S3 ) within the target gene (gene accession no: XM_015787560.1) employing CRISPR-P (Lei et al., 2014). The two selected sgRNAs were driven by rice-U6a and -U3 promoters and terminators, respectively, in the expression cassette ( Supplementary Figure S4A ) that then was cloned into the BsaI-BsaI restriction sites of the intermediary pMA-RQ entry vector. Eventually, the generated sgRNAs expression cassette was excised from pMA-RQ vector and cloned into the recipient NICTK-1_pCRISPR-Cas9 CRISPR-vector ( Supplementary Figure S4B ). Alongside, for knockin of the OsEPSPS-HDR template, HDR donor vector was developed, which encodes a mutated m-OsEPSPS polypeptide that harbors the amino-acid substitutions (G171A, T172I, and P177S) ( Supplementary Figure S4C ). BsaI restriction sites were added to the both left and right homology arm ( Supplementary Figure S5 ). Synthesized HDR template (1182) was cloned into the pMS-RQ entry vector.

The biolistic transformation was performed using Helium-powered Particle Delivery System PDS1000/He (Bio-Rad) with an acceleration pressure of 1,100 psi. The plasmid DNA of targeting- and donor-vector was extracted using a plasmid extraction kit (Thermo Fisher Scientific, India). Before bombardment, the concentration of DNA was optimized (2.0 µg/shot). Subsequently, we mixed the plasmid harboring the NICTK-1_pCRISPR-Cas9 vector with two gRNAs and purified PCR product of donor template that was amplified from pMS-RQ plasmid (harboring HDR template) in the molar ratio of 1:2. Embryogenic calli (30-day-old; 50–60 pieces) were bombarded using a protocol described by Kaul et al. (2021). After bombardment the, bombarded calli were transferred to MS-based regeneration media with varying hormonal supplementation for shoot regeneration. Regenerated shoots that adequately elongated were transferred to hormone-free ½-strength MS medium for rooting (Kaul et al., 2021).

To validate edited events, total genomic DNA (gDNA) was extracted from putatively transformed and wild-type (WT) rice leaves employing a modified Cetyltrimethyl ammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). The gDNA was utilized as template for PCR analysis. T₂ edited rice lines were validated by nested PCR analysis for Cas9 and OsEPSPS (mutated region) with specific primers (Agilent gradient thermocycler, Sure cycler 8800). PCR reaction conditions were optimized for each primer pair set ( Supplementary Table S1 ). Then, PCR product was visualized by gel documentation unit (Alpha imager EP), positively amplified DNA samples were gel-purified (QIAquick Gel Extraction Kit) and utilized for automated Sanger sequencing. The copy number of the Cas9 gene was confirmed by Southern blot analyses following methods described by Kaul et al. (2021). Approximately 10 μg of gDNA from the WT and Edited Lines (ELs) were digested with the EcoRV restriction enzyme. The digested products were size-fractionated on a 0.8% agarose gel and subsequently transferred onto N (+) nylon membranes. The blots were hybridized with a non-radioactively labeled PCR fragment (Cas9) probe in accordance with the instructions provided with the kit (Roche, Switzerland).

To investigate of glyphosate resistance level on seed germination phase, EL seeds of T₂ generation were germinated on ½-strength MS medium (Murashige and Skoog, 1962) harbored varying glyphosate concentration (0–4 mM/L). The germinated seeds were grown for 15 days in a glass jar under controlled culture conditions (25°C ± 2°C, with 16-h/8-h photoperiod and 2,000-lux intensity light/dark). WT seeds were grown at the same culture condition, i.e., with (0–4 mM/L) and without glyphosate, acting as positive and negative controls, respectively. Photographs were taken after 15 days of inoculation.

Furthermore, 30-day-old T₂ seedlings (10-leaf stage) were sprayed with up to a commercial glyphosate (6 ml/L; RR: 41.0% w/v; Monsanto Inc., Montreal, QC, Canada) under controlled greenhouse conditions (RH = 85%; Temp. = 28°C ± 2°C). The effect of glyphosate (appearance of any physiological abnormalities) was monitored regularly, and photographic evidence was recorded. The growth and yield potential of the ELs lines were also assessed. After glyphosate treatment (GT), 30-day-old seedlings were subsequently allowed to grow until maturity. At the maturity stage, agronomic traits with respect to flag leaf length and width, number of panicles per plant, panicle length, yield per plant, weight of 1000 grains, and number of tillers per plant were recorded.

Different photosynthetic parameters, i.e., net photosynthesis rate, photosystem II efficiency (Fv/Fm), intercellular CO₂ concentration, stomatal conductance, transpiration rate, and electron transport rate were measured from edited plants, TC, and WT on the third to fifth leaves from the top employed (Li-COR 6400–40, Lincoln, NE, USA). Conditions during the measurement were, i.e., photosynthetically active radiation (1,000 ± 7 μmol m⁻² s⁻¹ PAR: Parabolic aluminized reflector), humidity (79% ± 5%), temperature (24°C ± 2°C), and CO₂ concentration (400 μmol/mol). Abovementioned parameters were measured under light conditions, but except in the case of PSII quantum efficiency (Fv/Fm), which utilized dark-adapted (30 min) leaf. Data were recorded considering the relative leaf area in the leaf chamber. Experiments were repeated with three replicates.

Leaf strip bioassays were conducted to measure the injury caused by glyphosate application. Leaf segments (5 mm in size) were excised from both WT and edited plants and placed in a Petri plate. Each plate contains a minimum of six segments with 4.0 ml of either water (control) or solution with various concentrations (500-, 1,000-, 1,500-, 2,000-, 2,500-, 3,000-, 3,500- and 4,000 ppm) of glyphosate. The leaf strip containing petri plates that were placed under controlled culture conditions (25°C ± 2°C, with 16-h/8-h photoperiod and 2,000-lux intensity light/dark) was assessed after a 24-h interval, and the injury of the leaf (senescence) was recorded for each assessment. After 96 h of GT, the chlorophyll content was estimated followed by a protocol given by Arnon (1949). Briefly, leaf strips were grounded in liquid nitrogen and added 80% acetone (1 ml/100 mg of leaf tissue). The homogenate was centrifuged at 3,000g for 5 min, and the supernatant was collected to measure spectrophotometric absorbance (Thermo Scientific, India). The absorbance was quantitatively measured at 663 and 645 nm. On the basis of this absorbance, the concentration of chlorophyll-a (Chl a) and chlorophyll b (Chl b) was estimated using the following formulas:

EPSPS enzyme activity was examined via measuring released inorganic phosphate quantity employing the malachite green dye assay method (Lanzetta et al., 1979). The youngest fully expanded leaves (one- to two-tiller stage) of edited, TC, and WT were harvested after GT (0–10 mM), instantly frozen, and stored at −80°C. Marginal differences in optical density correlated positively with inorganic phosphate released; thus, EPSPS activity was measured in the forward direction. The reaction mixture consists with 1.0 mM S3P, 100 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), and 1.0 mM PEP. Total crude protein (2.0 mg) was obtained from edited ELs ER_1–6, WT, and TC after GT (0–10 mM) and made up to a final volume of 0.1 ml. After 20 min of incubation, the reaction was stopped by the addition of 1.0 ml of colorimetric solution (9.2 mM malachite green and 8.5 mM ammonium molybdate tetrahydrate in 1 M HCl, supplemented with 2 g of CHAPS/L to fix the color development) and 0.1 ml of 34% (w/v) sodium citrate solution after a minute. After 10 min of incubation, spectrophotometric absorbance (A660) was recorded (Thermo Fisher Scientific, India), the experiment was repeated with three replicates.

Leaf samples (youngest fully expanded) of six ELs, TC, and WT were harvested from the 30-day-old plant after 96 and 168 h of GT and utilized for SA quantification through High-performance liquid chromatography (HPLC) analysis following the reported protocol given by (Zelaya et al., 2011) with modification. Leaf samples (250 mg) were finely powdered in liquid nitrogen and then centrifuged at 20,000g for 15 min to separate plant debris components, and the collected plant extracts (10–30 μl) were filtered (0.22-μm nylon membrane) and were analyzed by HPLC employing Millennium software (Waters Corp., Milford, MA). The separation was done via utilizing an analytical column LiChrosorb NH2 (Phenomenex, Torrance, CA) with a flow rate of 1 ml/min of mobile phase [95% acetonitrile + 5% (4:1 water:orthophosphoric acid)]. For good linearity confirmation, the chromatograms were performed at 210 cm⁻¹ with a retention time of 2.35 min. The Level of detail (LOD) and Limit of quantification (LOQ) values were determined at a noise value of 3 and 10, respectively. Shikimic acid elution was observed at 210 nm, resulting in an Room temperature (RT) of 7.1 min. Shikimic acid standard curve is also prepared to utilize commercially available shikimic acid (>99% pure, Sigma-Aldrich). The experiment was assayed in three replicates.

To quantify the aromatic amino acid content, the leaf samples from ELs, WT, and TC were finely powdered and dissolved in a mixture of acetonitrile and ammonium formate (80:20) after 96 h of RR foliar spray utilizing liquid chromatography–mass spectrometry (LC-MS) system (Thermo ScientificTM TSQ FortisTM) equipped with Thermo Trace finder software and positive ion mode electrospray ionization. Supernatant (0.5 µl) was used to quantify amino acid (Phe, Trp, and Tyr). LC separation was done utilizing Buffer A (50 mM ammonium formate) and Buffer B (0.1% formic acid in acetonitrile). The LC flow rate was fixed at 0.05 ml/min. The LC gradient increased (30% to 90%) in 10 min at mobile phase B, held for 5 min at 95% B, returned in 1 min to 30% B, and then held for 6 min at 30% B (Ibsen et al., 2013). Following the LC as mentioned above conditions, WT, TC, and ELs samples were separately eluted from the LC column with baseline separation at an RT (10 mg/ml in 0.1 M HCl) of standards Phe, Trp, and Tyr.

Pollen viability was examined using 2% aceto-carmine solution as described by Rathod et al. (2018). Thirty-day-old seedlings of edited rice lines were foliar sprayed with RR (6 ml/L), and, upon maturity, pollen was collected to check their viability. Pollen from the WT plant (without RR sprayed) is considered a positive control. Collected pollen from dehiscing anthers of both edited, and WT plants were placed on slides with aceto-carmine solution, and photographs were taken using microscopy (Nikon). Pollens with red color were considered as viable and colorless one as nonviable.

All experiments were conducted in triplicate with three independent biological replicates. Data were analyzed statistically via one-way analysis of variance (ANOVA) using a complete randomized design. The groups that showed variance were then subjected to Tukey HSD test (HSD_0.5) and Duncan’s Multiple-Range Test 10 with a significance value of p ≤ 0.05.

GR weed biotypes were generated due to the intense selective pressure of continuous, heavy glyphosate use. Homology searches, phylogenetic analysis, and amino acid alignment of EPSPS encoding genes from various crop plants revealed a highly conserved PEP-binding domain. Continuous application of glyphosate herbicide invoked a selection pressure that led to natural spontaneous introduction of favorable mutations (TIPS) in the conserved motif that represented the PEP-binding active site of the target enzyme EPSPS, which, in turn, conferred resistance to glyphosate in monocot, dicots, and bacteria ( Figure 1A ). At the protein level, OsEPSPS shared 76%–89% sequence identities with other class I plant EPSPSs and 21% with class II EPSPS genes, for instance, found Agrobacterium sp. strain CP4 ( Figure 1B ). In silico sequence analysis of OsEPSPS proteins exhibited conserved amino acid residues, i.e., G172, T173, and P177, that played a crucial role as might function as the PEP-binding catalytic domains of EPSPS enzymes in monocots and dicots. As the PEP and glyphosate showed analogous structures, hence glyphosate mimicked PEP and competitively inhibited it from binding to the active site within EPSPS ( Figures 1C–E ). Hence, GATIPS amino acid substitution in PEP-binding site motif via CRISPR-Cas9 approach may lead to the development of GR rice plant.

To date, no reports exist, wherein cultivated crops have revealed any spontaneous or induced GATIPS triple mutations, specifically due to reduced probability of three concomitant nucleotide substitutions/replacements. In line with the above, we introduced GATIPS amino acid substitution mutations in the native OsEPSPS gene ( Supplementary Figures S2 , S3 ) employing CRISPR-Cas9–based homologous recombination. We utilized a robust marker-free pCAMBIA1300–based binary vector NICTK-1_pCRISPR-Cas9 (16.0 kb) that harbored the rice codon-optimized SpCas9 gene for expressing sgRNAs in rice (monocots). The Cas9 gene expression cassette comprised the coding sequence of Cas9 gene flanked on both sides NLS. In addition, the Cas9 gene expression was driven by a maize ubiquitin (pZmUbi) promoter along with NOS terminator ( Figure 2A ; Supplementary Figure S4A ). Two gRNAs were designed to target the OsEPSPS gene employing CRISPR-P software (Lei et al., 2014). We earmarked two rice-U6a and -U3 promoters that facilitated the expression of two-sgRNA cassettes and resulted in DSBs within the regions that flanked the conserved PEP-binding (target) site in native OsEPSPS in rice ( Figure 2B ). The designed sgRNA cassettes were synthesized and cloned into the entry vector pMA-RQ using BsaI-BsaI restriction digestion and ligation. The integration of sgRNA expression cassettes was verified by nested PCR and sequencing analyses. Finally, the generated sgRNA expression cassettes were cloned into one recipient pCAMBIA1300-based indigenously developed NICTK-1_pCRISPR-Cas9 vector that ultimately generated the OsEPSPS_NICTK-1_pCRISPR-Cas9 construct for transformation in rice ( Supplementary Figure S4B ). Furthermore, the integration of sgRNA cassettes was verified by nested PCR and sequencing analyses.

Rationalized designing of HDR templates led to enormously improved HDR efficiencies in CRISPR-based genome editing experiments. To develop GR rice plant, an HDR donor vector was generated ( Supplementary Figure S4C ), which encoded a mutated m-OsEPSPS polypeptide that harbored the amino-acid substitutions (G171A, T172I, and P177S) ( Figure 2C ). Sequences corresponding to desired edits were positioned in the middle of the HDR template. Sequences (~500 bp) immediately after upstream and downstream of the target insertion sites were selected as 5′- and -3′ homology arm, respectively. Moreover, we introduced a silent mutation in PAM sequence to prevent undesired point mutation within the gene ( Supplementary Figure S5 ). Synthesized HDR template (1,182) then was cloned into the BsaI-BSaI restriction sites of the intermediary pMS-RQ entry vector.

Efficacious introduction of stable mutations into rice genome employing CRISPR-Cas reagents (Kaul et al., 2019; Kaul et al., 2021) via biolistic approach (Kaul et al., 2021; Kaul et al., 2022) has been previously reported. We co-delivered the vector OsEPSPS- NICTK-1_pCRISPR-Cas9 and the PCR-amplified OsEPSPS-HDR template into rice calli via biolistic approach (Li et al., 2016) ( Figure 2D ) to develop GR ELs that harbored three mutations within the PEP-binding site of the native EPSPS gene. The transgene integration was verified by PCR analysis using Cas9 gene-specific primers ( Figure 2E ; Supplementary Table S1 ). A total of 1,600 embryogenic rice calli were bombarded, and, eventually, we obtained 1,059 putative Cas9-positive plants ( Supplementary Figures S6A, B ). Subsequently, the putative T₂ ELs were validated by nested PCR and Sanger’s sequencing analyses by mOsEPSPS gene-specific screening primers (including the mutated nucleotides) for identification of the desired mutations. Ten out of the selected 66 Cas9(+) plants exhibited the putative integration of the donor template ( Figures 2F–H ). Hence, nested PCR-positive ELs that were eventually confirmed via Sanger sequencing revealed the incorporation of requisite three–amino acid replacements (G172A, T173I, and P177S) in the native OsEPSPS gene. The sequence chromatograph revealed the homozygosity of the developed ELs ( Figures 3A, B ). The Southern blot analysis performed employing Cas9 gene-specific probe exhibited the number of Cas9 gene copies integrated into the genomes of T₁ lines as reflected by the corresponding signals ( Figure 2I ). Whereas, the lanes in the blot that incubated the digested gDNA of the non-transformed plant/WT revealed an absence of signal. Six out of the 10 independent rice T₁ edited lines that showed better performances were selected for agronomic trait analyses.

Finally, to scrutinize the Cas9-free edited T₂ rice lines, we performed PCR assays of individually selfed T₂ plants using Cas9-specific primers. Among them, 60 T₂ (10 plants per line) were selected for PCR analysis, and, out of these, seven lines failed to generate an amplicon of the expected size ( Figures 4A–G ; Supplementary Figure S7 ). These putative Cas9-free plants were further validated by Southern blot analysis, and the hybridized blot revealed seven Cas9-free T₂ plants ( Figure 4H ). Results indicated that these Cas9-free plants carrying the targeted gene stably transferred three mutations in OsEPSPS to subsequent generations and exhibited indistinguishable phenotypes with no yield penalties ( Figure 4I ).

To determine the glyphosate-induced foliar damage in the ELs, chlorophyll assay was performed. Leaf strips of ELs and WT were incubated with different glyphosate concentrations (10³ to 4 × 10³ ppm). After 48 h of incubation in varying concentrations of glyphosate, the leaf strips of ELs retained more chlorophyll content than their counterparts (WT and TC), hence revealing relatively lower levels of chlorophyll degradation. Under control conditions, negligible differences were observed in total chlorophyll (Chl) contents in ELs in comparison to WT. Previously, with regard to foliar spraying assay, we had observed that glyphosate-induced damage was relatively lower in ELs as compared to WT and TC. The results of leaf-strip assay were in line with and exhibited similar performances as recorded during the foliar spraying assay. The ELs showed minimal leaf senescence rate that, in turn, was reflected as higher chlorophyll contents in edited plants ( Supplementary Figures S10A, B ). In the presence and absence of glyphosate, the chlorophyll contents of WT, TC, and ELs were comparable at 2,500 ppm. Degradation of cellular chlorophyll content started at 3,000 ppm in TC plants, whereas the complete degradation was observed at 4,000 ppm. On the other hand, ELs retained higher chlorophyll contents in comparison to TC treated with 4,000 ppm, similar to WT. Thereby, the results collectively implied that GR rice ELs displayed minimal deterioration of photosynthetic pigment, which, in turn, facilitated their survival on application of herbicide (up to 4,000 ppm).

The EPSPS enzymatic activity was assayed by quantifying the inorganic phosphate released at the time of EPSP formation from PEP and S3P. The malachite green dye assay was performed to analyze the kinetic parameters of the OsEPSPS protein in edited rice lines, wherein the estimated optical density was plotted on a graph ( Figure 6A ). The assay was conducted in the presence of varying concentrations of glyphosate (0–10.0 mM/L) for crude or unrefined protein samples (6 ELs, WT, and TC). In case of WT, the OsEPSPS enzymatic inhibition rate concomitantly incremented with the increased glyphosate concentrations and, eventually, at 6.0 mM/L, the OsEPSPS activity was entirely restrained. Interestingly, in three ELs (ER_2,4,6), complete inhibition was observed at 10.0 mM/L. However, even at 10.0 mM/L, the OsEPSPS activity was not completely restrained in the case of ER_1,3,5. Hence, edited lines ER_1,3,5 revealed higher resistance against glyphosate than ER_2,4,6. On the basis of this assessment, we inferred that ELs were significantly tolerant to glyphosate than WT.

The glyphosate-induced injury may be assessed by estimation of shikimate levels within a crop. Glyphosate occupies the PEP-binding active site within OsEPSPS protein, thereby successfully shutting down the shikimate biosynthesis pathway via restraining the EPSPS activity, and, in the process, leads to the accumulation of the precursor, shikimic acid (SA), and reduction in the end products, i.e., aromatic acids. The quantitative estimation of SA content was performed employing HPLC analysis. In general, SA accumulation started after 24 h of treatment, and, after 96 h, we observed notable differences in SA levels. We quantified the SA contents in edited T₂ and WT rice lines along with TC samples after they were sprayed with RR. Samples were collected after 4- and 7-day time points and subjected to HPLC analysis. SA contents of six ELs, WT, and TC lines were measured by comparing their retention times with the commercially available standard SA solution (99% Purity; Sigma, India) that was resolved adequately to give baseline separation. Varying concentrations (10) of the standard were injected in triplicate, and an average peak area was projected. We observed that, in the TC rice line (RR foliar spray treatment), there was a concomitant enhancement in shikimate concentration with time. However, edited lines revealed low levels of SA, similar to the WT. For instance, after RR treatment, TC rice line revealed a 2.4-fold enhanced accumulation of SA than WT. On the contrary, edited T₂ rice lines (ER_1–6) revealed lower levels of endogenous shikimate (14.5-fold) in contrast to TC but quite similar to WT following treatment with glyphosate ( Figure 6B ).

EPSPS is the sixth essential enzyme in the biosynthetic pathway of aromatic amino acids in the plants. To find the effect of gene-editing of EPSPS on the synthesis of aromatic amino acids (Phe, Trp, and Tyr) in ELs, we measured the levels of Phe, Trp, and Tyr in WT, TC, and ER lines employing the LC-MS approach. Interestingly, we observed that, after RR foliar spraying, the Phe, Trp, and Tyr contents significantly increased in ER lines in comparison to WT. The ER T₂ lines revealed significantly increased levels of aromatic amino acids in comparison to WT. The GT led to a two-fold increase in Phe, 2.5-fold increase in Trp, and two-fold increase in Tyr contents in ELs as compared to WT ( Figure 6C ). Overall, the results indicated that altered EPSPS enzyme activity revealed significantly high levels of Phe, Trp, and Tyr in the ELs after RR (glyphosate) foliar application (6 ml/L) and displayed enhanced resistance to the herbicide as compared to WT. Only specific amino acid profiles associated with only the shikimate pathway were affected with no significant changes on the total free amino acid. The TC showed increased aromatic amino acid content after RR foliar treatment (6 ml/L), but these lines eventually died.

We concluded that the edited rice lines (ELs) harboring the GATIPS amino acid substitutions employing CRISPR-Cas9 approach exhibited significantly enhanced levels of glyphosate-resistance, even at a high dosage of RR foliar applications (6 ml/L). Conjunctly, Cas9-free T₂ ELs revealed a high accumulation of essential aromatic amino acids via more efficient utilization of the shikimate. The ER lines were phenotypically indistinguishable from WT with no yield penalties ( Supplementary Figure S11 ).