Genome Wide Evaluation of the Impact of Targeted Gene Editing in Diploid Potato

Genetically engineered (GE) crops are a key component of U.S. agriculture and quantitative data to facilitate informed risk assessment of GE crops is important. Aim of this project is to utilize the targeted genome editing technologies, Transcription Activator–like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR-cas) to engineer a crop species and detect the variability generated at the genomic level compared to non-GE approaches.

A diploid inbred potato line 523-3 (M6) (S. chacoense 2n=2x=24) is used to engineer for herbicide resistance by modification of the endogenous acetolactate synthase (ALS) gene.  Specific mutations in ALS are known to confer resistance to sulfonylurea and imidizolinone herbicides. The genome of M6 was sequenced and assembled with an assembly size of 826 Mb, N50 scaffold of 715 kb. CRISPR/Cas reagents developed expressing single-guide RNA targeting the StALS1 gene successfully induced targeted mutations in other diploid and tetraploid potato lines using Agrobacterium-mediated transformation. TALENs reagents that included a repair template designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus successfully integrated supporting herbicide resistance phenotype.

Herbicide resistance will be generated using Ethyl methanesulfonate (EMS) and gene targeting reagents in M6. Genome wide identification of DNA double stranded breaks induced by the nucleases will be analyzed by sequencing using GUIDE-seq approach to detect the off-target sequences. Whole transcriptome profiling to assess the impact on expression patterns and agronomic phenotypes will be analyzed in the field. This data will quantify the potential of off-target effects in GE versus non-GE crops.