CRISPR/Cas-mediated base editing of the lettuce acetolactate synthase gene

BERACOCHEA, VALERIA; RADONIC, LAURA M.; MARISA LOPEZ BILBAO; DARQUI, FLAVIA S.; HOPP, H. ESTEBAN

Modalidad virtual

Simposio; Keystone Symposia eSymposia meeting Plant Genome Engineering: From Lab to Field | EK25; 2021

Keystone Symposia

In different plant species, specific mutations in the acetolactate synthase gene (ALS) lead to amino acid substitutions that confer herbicide resistance. The spontaneous substitution of Pro197 for His in the ALS gene of L. serriola (wild lettuce) generated resistance to sulfonylureas and imidazolinones. This characteristic could be transferred to cultivated lettuce (L. sativa) by crossing. Later, substitutions of Pro197 for Thr, Ser and Leu were detected in herbicide-resistant wild lettuce plants.Our objective is to replace the Pro197 (CCC) of the L. sativa ALS gene (LsALS) by Ser (TCC/TCT) or Leu (CTC / CTT) through base editing, in order to obtain herbicide-resistant lettuce lines. This will allow us to optimize the gene editing methodology and the selection of herbicide-resistant events in our laboratory. The present work describes the assembly of the editing vector, its delivery by genetic transformation and the evaluation of T0 and T1 plants.We identified the target region in the LsALS gene through PCR amplification and sequencing and we verified the absence of allelic variants. For the assembly of the editing vector, we used the plasmid pXSE901BG (Addgene) and replaced the glyphosate/glufosinate ammonium resistance cassette with a kanamycin resistance cassette and we incorporated the gRNA-spacer sequence targeting LsALS-Pro197. We transformed 132 lettuce cotyledons (var. Grand Rapids), obtaining 8 different transformation events. From each kanamycin-resistant calli, we obtained 1 to 3 T0 plants. Genomic DNA was extracted from T0 adult plant leaves. We confirmed transgenesis through PCR amplification and sequencing of different T-DNA fragments. However, in these samples, we did not detect any C by T changes in the target region. From the self-pollination of these T0 plants, we obtained T1 seeds. T1 plants showed different segregation patterns of the kanamycin-resistance phenotype. At the moment, we are adjusting an in vitro system for the detection of putative-edited events, by germinating T1 seeds in half strength MS medium supplemented with the sulfonylurea herbicide chlorsulfuron.