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

ABSTRACT The 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 21 st century. 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 an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA prior to in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity 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 high in vitro cleavage activity did not produce edits in vivo , suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage 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 observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure in vivo cleavage of chosen sgRNAs. Key Message in vitro cleavage assay of sgRNAs was able to identify low activity sgRNAs but did not 13 reliably predict in vivo mutagenesis. Using multiple sgRNAs that meet the plant-validated parameters and have high activity in vitro in plant genome editing is critical to ensure success.