New Insights into CRISPR-Cas9 sgRNA Design for Plants

One of the challenges of using CRISPR-Cas9 in plants is designing single guide RNAs (sgRNAs) that can accurately target the desired gene. Many different sgRNA design tools are available, but many are based on guidelines designed for animals. Researchers from the University of the Philippines Los Baños investigated the in vitro and in vivo activity of four different sgRNAs targeted for several eggplant polyphenol oxidase gene family members. They found some sgRNAs with high in vitro cleavage activity did not produce edits in vivo. This suggests that an in vitro assay may not be reliable for predicting sgRNAs with highly efficient in vivo cleavage activity.

Other sgRNA design algorithms were also analyzed, including those considering plant-specific factors such as secondary structures and base-pairing. They found these algorithms could help explain the discrepancy between their observed in vitro and in vivo cleavage efficiencies. However, the researchers also note that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs. This is because many factors can affect sgRNA cleavage efficiency, such as off-target sites in the genome and the delivery method used to deliver the CRISPR-Cas9 system into the plant.

The researchers concluded that it is essential to consider the sgRNA design tools and algorithms used carefully and to validate sgRNA cleavage efficiency in vivo before proceeding with further experiments.

Read the abstract in Transgenic Research.