The herbicide tolerant canola (Brassica napus) event GE-CLB1 was developed using site-directed mutagenesis of the ahas3 gene in B. napus (A genome). This gene encodes an acetohydroxyacid synthase (AHAS) enzyme, also referred to as an acetolactate synthase (ALS) enzyme.

Gene repair oligonucleotides were introduced into protoplasts of conventional spring oilseed rape line BN02 using a PEG treatment, introducing two targeted mutations in the ahas3 gene. These point mutations result in an alanine to threonine substitution at position 122 (A122T) and a serine to asparagine substitution at position 653 (S653N) and render the modified AHAS3 enzyme insensitive to imidazolinone herbicides, including imazamox and imazapyr.

Sequencing analyses confirmed the presence of the two intended point mutations and the absence of any mutations in the closely related ahasL1C gene. PCR results demonstrated the stable presence of the GE-CLB1 trait across multiple generations. Segregation data confirmed the stability of the trait, showing that it segregates in a Mendelian fashion.