Development of a CRISPR/SHERLOCK-Based Method for Rapid and Sensitive Detection of Selected Pospiviroids

Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.