CRISPR-Cas9 mediated genome editing for developing herbicide-tolerant rice lines

Abstract:

CRISPR-Cas9 is a programmable nuclease system that enables precise, targeted modifications in plant genomes without introducing foreign DNA into the final product. This research applied CRISPR-Cas9 to edit three herbicide-target genes—ALS, ACCase, and EPSPS—in indica rice (cv. Pusa Basmati 1) to develop lines tolerant to imazethapyr, quizalofop, and glyphosate, respectively. Four sgRNA constructs were designed, cloned into pCas9 vectors, and delivered via Agrobacterium-mediated transformation at the Chandra Shekhar Azad University of Agriculture, Kanpur, India, during 2021–23. Regeneration efficiency ranged from 26.4% (pCas9-EPSPS) to 34.2% (pCas9-ALS1). Among 186 T₀ plants screened, 47 carried confirmed edits at the target loci. T₁ segregation analysis identified 12 homozygous edited lines. Herbicide tolerance assays in T₁ showed that edited line CR-15 (ALS double mutant) tolerated imazethapyr at 4× the field rate without visible injury, scoring 4.8 on a 5-point tolerance scale versus 1.8 for the wild type. No off-target mutations were detected at the top five predicted sites. Agronomic performance of the best edited lines was statistically equivalent to the parent variety. These results demonstrate that CRISPR-based point mutations in herbicide-target genes can produce non-transgenic, herbicide-tolerant rice suitable for direct-seeded cultivation systems.