White spot disease (WSD) caused by white spot syndrome virus (WSSV) continues to be a major threat to shrimp farming worldwide. Despite major progress in characterizing the virus, developing tools for WSD diagnostics and understanding in WSSV pathogenesis, no therapeutic is available to control WSD. This project was designed to test the hypothesis that CRISPR-Cas9 directed genome editing can be used as a therapeutic to combat the progression of WSD in live shrimp. Multiplexed CRISPR-Cas9 constructs were made such that the functional components of the CRISPR-Cas9 system would be expressed in shrimp. The multiplexed vectors had three specific guide RNAs that targeted three sites in three different WSSV genes, VP28, DNA polymerase, and ribonuclease reductase 1. In laboratory challenge experiments, Penaeus vannamei shrimp were injected with these plasmid constructs either singly or in a combination mixture of plasmid DNA. Once shrimp were injected with the genome editing vectors, they were orally challenged with WSSV. An arbitrary amount of DNA was used for injection modeled on prior studies using RNAi vectors in other systems. The expectation that this would impact the progression of the disease was low, but the idea was to look for any edited WSSV genes in moribund or dead shrimp to look for genome editing. Surprisingly, most of the CRISPR-Cas9 treated shrimp had a delayed onset of mortalities compared to the positive control. We are currently analyzing the samples for the detection of predicted editing in the targeted WSSV genes.
While these were preliminary experiments, it was surprising to see any whole animal impact of the direct genome editing of an invading virus - this is unique to the field. These experiments need to be confirmed and the dose and delivery of the DNA based therapeutic refined to have a positive impact in developing a commercially viable therapeutic for WSD control.