CRISPR-BASED DIAGNOSTICS FOR RAPID DETECTION OF WHITE SPOT SYNDROME VIRUS IN SHRIMP

The pacific white shrimp Penaeus vannamei is the most profitable crustacean aquaculture organism globally. Production of white shrimp has quadrupled since 2000 and sits at an all-time high, with an estimated yearly value of ~12-15 billion dollars. While productivity is high, impediments to increased supply continue to impact shrimp aquaculture. One of the greatest impediments is the spread of diseases. Pacific white shrimp have a long history of issues related to infectious viral diseases, from Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) which emerged in 1981, Yellow Head Virus (YHV) and Taura Syndrome Virus (TSV) which emerged in 1991, Infectious Myonecrosis Virus (IMV) emerging in 2004 and White Spot Syndrome Virus (WSSV - one of the most influential of all viral epizootics) which emerged in 1992. The impact these diseases have had is best exemplified in the costs they have taken on global shrimp production, estimated at well over 20 billion dollars. WSSV itself has cost over 6 billion dollars since its emergence due to large scale rapid mortality events around the world.

Due to the rapid progression of WSSV and the inability to vaccinate crustaceans, early diagnostics are critical for limiting transmission. However, current diagnostics using histology, PCR, qPCR, and RT-PCR require sophisticated equipment, expensive reagents, specialized training and are time intensive (~ days to a week), which is outside the window where action can be taken. In contrast, we are proposing to develop new next-generation CRISPR technologies (i.e. SHERLOCK - Specific High sensitivity Enzymatic Reporter UnLOCKing) to the diagnosis WSSV infecting shrimp. This new technology will allow for diagnostic assays that are inexpensive, rapid (~1-3 hours), extremely sensitive (attomolar level), require no complex equipment or specialized training, and can be employed in the field or at the pond-site. This will enable rapid and effective management to safeguard aquaculture production by ensuring the production of genetically superior, disease free broodstock and post-larvae (PL) through captive breeding programs, by limiting movement of infected broodstock and PL, and increasing biosecurity.

We designed and evaluated a number of primers and guide RNA probes for the amplification of WSSV DNA and detection of WSSV sequences. Trials with these assays resulted in an assay that was able to detect down to 10 synthetic viral DNA copies with strong linearity in our standard curve (Fig. 1). Tests of previously challenged samples showed high accuracy and specificity, only detecting infected samples, and not detecting other OIE-listed epizootics (TSV, IHHNV, YHV, etc). Finally, a comparison of our assay to qPCR showed the SHERLOCK assay had sensitivity down to ~1-10 copies, and that both assays were highly congruent for the quantification of viral load from unknown challenged samples. We are currently evaluating the robustness of our method by testing a large number of individuals.