Usefulness of Current sgRNA Design Guidelines andin vitroCleavage Assays for Plant CRISPR/Cas Genome Editing: A Case in Eggplant (Solanum melongenaL.)

Abstract

ABSTRACTThe advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed on the development of new crop varieties that can withstand agricultural challenges of the 21stcentury. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to a multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether anin vitroassay is needed to validate the targeting efficiency of a particular sgRNA prior toin vivodelivery of the CRISPR/Cas9 system. Here, we demonstrate thein vitroandin vivoactivity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have highin vitrocleavage activity did not produce editsin vivo, suggesting that anin vitroassay may not be a reliable basis to predict sgRNAs with highly efficientin vivocleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observedin vitroandin vivocleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensurein vivocleavage of chosen sgRNAs.Key Messagein vitrocleavage assay of sgRNAs was able to identify low activity sgRNAs but did not 13 reliably predictin vivomutagenesis.Using multiple sgRNAs that meet the plant-validated parameters and have high activityin vitroin plant genome editing is critical to ensure success.