In Silico Prediction and Analysis of Potential Off-Targets and Off-Target Mutation Detection in StERF3-Gene Edited Potato Plants

The imperative aspect of the CRISPR/Cas9 system is a short stretch of 20 nucleotides of gRNA that control the overall specificity. Due to the small size, the chance of its multiple occurrences in the genome increases; however, a few mismatches are tolerated by the Cas9 endonuclease activity. An accurate and careful in silico-based off-tar-get prediction while target selection is preferred to address the issue. These predictions are based on a compre-hensive set of selectable parameters. Therefore, we investigated the possible off-target prediction and their screening in StERF3 gene-edited potato plants while developing StERF3-loss-of-function mutants using CRISPR/Cas9 approach. The 201 off-targets for the selected targets of the StERF3 gene were predicted, and 79 were filtered as potential off-targets. Of these 79, twenty-five off-targets showed scores with defined cut-off values <0.5 and were analyzed in Sterf3-edited potato plants compared to wild-type plants. No off-targeting was found to have occurred in edited plants.