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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 phosphoenolpyruvatebinding site in the native OsEPSPS gene employing fragment knockout and knockin 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 glyphosateresistance 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 treatedWT 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. [ABSTRACT FROM AUTHOR]

Genome engineering by site-specific nucleases enables reverse genetics and targeted editing of genomes in an efficacious manner. Contemporary revolutionized progress in targeted-genome engineering technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-related RNA-guided endonucleases facilitate coherent interrogation of crop genome function. Evolved as an innate component of the adaptive immune response in bacterial and archaeal systems, CRISPR/Cas system is now identified as a versatile molecular tool that ensures specific and targeted genome modification in plants. Applications of this genome redaction tool-kit include somatic genome editing, rectification of genetic disorders or gene therapy, treatment of infectious diseases, generation of animal models, and crop improvement. We review the utilization of these synthetic nucleases as precision, targeted-genome editing platforms with the inherent potential to accentuate basic science "strengths and shortcomings" of gene function, complement plant breeding techniques for crop improvement, and charter a knowledge base for effective use of editing technology for ever-increasing agricultural demands. Furthermore, the emerging importance of Cpf1, Cas9 nickase, C2c2, as well as other innovative candidates that may prove more effective in driving novel applications in crops are also discussed. The mined data has been prepared as a library and opened for public use at www.lipre.org. [ABSTRACT FROM AUTHOR]

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native Zm EPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-m Zm EPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-m Zm EPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the Zm EPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize. • Glyphosate, an ingredient of Roundup herbicide, inhibits EPSPS, a key enzyme of the aromatic amino acid biosynthetic pathway. • CRISPR/Cas9- and HDR-based editing of ZmEPSPS enabled triple amino acid substitutions (GATIPS) to inhibit glyphosate binding. • ZmEPSPS editing conferred high yield and enhanced glyphosate resistance in maize plants. • This is the first report to employ a CRISPR/Cas9- and HDR-based approach to generate herbicide resistant maize plants. [ABSTRACT FROM AUTHOR]