Genome Editing in Lettuce

One focus of the Michelmore lab is the identification and manipulation of disease resistance genes in lettuce using contemporary genetics and genomics technologies. The lettuce genome has been sequenced for multiple genotypes and more than 52 phenotypic loci that confer resistance to ten diseases have been mapped. Disease resistance genes tend to be located in major resistance clusters that contain many similar genes encoding nucleotide binding site and leucine-rich repeat proteins. Cloning specific resistance genes is therefore challenging. We have used RNAi to assign resistance phenotypes to subsets of genes within families but not to individual causal genes. We are using a pooled gRNA CRISPR strategy that aims to target all genes in a specific R-gene family. To demonstrate the efficiency of generating monoallelic and biallelic gene knockouts in lettuce with CRISPR, we targeted a gene involved in seed germination thermotolerance (NCED4), which provides a whole-plant selectable phenotype. Two lettuce genotypes were transformed with three different Ubi:CAS9 constructs each containing one gRNA targeting NCED4. This resulted in seeds that could germinate at elevated temperatures that were non-permissive for the wildtype. A second generation of edited plants is underway. In the longer term, we are investigating editing approaches that do not involve tissue culture. We will also develop gene targeting to create stacks of resistance genes containing several R genes at a single Mendelian locus. This will enhance the durability of resistance by increasing the evolutionary hurdle that the pathogen will have to overcome and greatly simplify breeding for disease.