CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.).

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 ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) 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-mZmEPSPS 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 ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Phylogenetic and modelling analysis of purple acid phosphatase 18 (SiPAP18) from Setaria italica.

Purple acid phosphatases belong to metallo-phosphatase family. Intracellular phosphatases are crucial for phosphorus (P) distribution in the cell and are highly induced in phosphorus-deprived conditions in the soil. Disparate PAP isoforms exist within discrete subcellular compartments in Setaria italica and their expression in P deprived conditions fosters phosphorus amelioration. We isolated the SiPAP18 gene and developed the homology SiPAP18 protein model based on the crystal structure of the Kidney bean PvPAP (PDB ID: 2QFP) as template (sequence similarity 42.7%) using Modeller 9.12 with adequate validation. Structure model analysis shows the significance of five conserved signatures with seven metal-paired amino acid residues during P-deprivation induced phosphorus amelioration.