Crispr-cas9 genome editing for improving nitrogen and water use efficiency in wheat

Overview

Wheat production globally consumes roughly 18% of nitrogen fertilizer and 108 billion cubic meters of water annually, making input efficiency a critical priority for food security and farmer profitability. This project applies SDN1-mediated CRISPR-Cas9 genome editing to knock out two key regulatory genes, ARE1 and DST, in elite Indian wheat cultivars to simultaneously improve Nitrogen Use Efficiency (NUE) and Water Use Efficiency (WUE).

ARE1 is a negative regulator of nitrogen assimilation; its inactivation in rice and wheat has been associated with delayed senescence, 10–20% grain yield increases, and improved NUE. DST is a zinc-finger transcription factor linked to drought and salt tolerance; loss-of-function edits in rice have improved WUE and reduced crop water requirements by approximately 25%. Because India's regulatory framework exempts transgene-free CRISPR-Cas9 knockout mutants from GMO oversight, successfully edited lines can move rapidly toward commercial cultivation.

Technical specifications

Target genes and traits:

• ARE1 knockout: targets a negative regulator of nitrogen assimilation to enhance NUE and grain yield
• DST knockout: targets a zinc-finger transcription factor to improve drought tolerance and WUE

Editing platform and methodology:

• SDN1 CRISPR-Cas9 approach producing transgene-free edited lines
• sgRNAs designed using WheatCRISPR software and cloned into the pDIRECT_25F binary vector
• Validated Agrobacterium-mediated transformation protocol for Indian wheat genotypes (DBW187, DBW303) established by the team
• Established genotype-independent, highly reproducible regeneration system

Validation pipeline:

• Sanger sequencing of target regions in T0/T1 edited plants to confirm edits
• Cas9 and hygromycin marker screening to identify transgene-free T2 lines
• Allele characterization using DSDecode and CRISP-ID decoding tools
• Phenotypic evaluation of homozygous T2 lines for agronomic, yield, NUE, and WUE traits under greenhouse and field conditions

Technology readiness level

The project builds on a fully standardized Agrobacterium-mediated transformation and regeneration system for Indian wheat genotypes, with prior successful CRISPR-Cas9 edits of GW2 and MS1 genes already advanced to the T2 generation in cultivar DBW187. Cas9-free promising lines have been identified in earlier work. The current effort is at the T0/T1 generation stage for ARE1 and DST edits, with molecular and phenotypic characterization underway. Given India's regulatory exemption for transgene-free CRISPR knockouts, successfully validated lines are positioned for relatively rapid farmer deployment following field trials.