Recipient Organization
SHERLOCK BIOSCIENCES, INC.
200 TALCOTT AVE
WATERTOWN,MA 02472
Performing Department
(N/A)
Non Technical Summary
Rapid and accurate health assessment of farmed animals is critical to US aquaculture and agriculture. It is hindered by the cost, time, and technical expertise required for standard diagnostic methods, resulting in reduced capacity for disease control and production losses. Our goal is to transform US animal production by developing rapid, inexpensive, sensitive, field-deployable CRISPR-based diagnostics that enable farmers to effectively monitor and respond to disease outbreaks, validate pathogen-free status of broodstock, and screen imported materials for threats. The target pathogen for this Phase I project is White Spot Syndrome Virus (WSSV), a devastating virus that disrupts shrimp farm operations worldwide, resulting in billions of dollars in lost production. WSSV is highly virulent, leading to mass mortality such that early, rapid detection is critical to prevent catastrophic losses and mitigate spread. The specific objectives of this project are to (1) advance an established WSSV CRISPR-based diagnostic assay to be compatible with a cartridge-based diagnostic device ("PowerLite") developed by Sherlock Biosciences, (2) develop a simple field-based sample collection procedure compatible with the CRISPR detection cartridge (3) test the field deployable WSSV cartridge on the "PowerLite" prototype. This innovative technology would improve biomonitoring practices by enabling shrimp farmers to more rapidly and accurately screen for, and respond to, WSSV. It would provide a platform that could extend to other animal pathogens that impact US aquaculture and agriculture. This provides a unique business opportunity for Sherlock Biosciences to expand its technology platform beyond human health to domestic and global food security.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
Rapid and accurate health assessment of farmed animals is critical to US aquaculture and agriculture. It is hindered by the cost, time, and technical expertise required for standard diagnostic methods, resulting in reduced capacity for disease control and production losses. Our goal is to transform US animal production by developing rapid, inexpensive, sensitive, field-deployable CRISPR-based diagnostics that enable farmers to effectively monitor and respond to disease outbreaks, validate pathogen-free status of broodstock, and screen imported materials for threats. The target pathogen for this Phase I project is White Spot Syndrome Virus (WSSV), a devastating virus that disrupts shrimp farm operations worldwide, resulting in billions of dollars in lost production. WSSV is highly virulent, leading to mass mortality such that early, rapid detection is critical to prevent catastrophic losses and mitigate spread. The specific objectives of this project are to (1) advance an established WSSV CRISPR-based diagnostic assay to be compatible with a cartridge-based diagnostic device ("PowerLite") developed by Sherlock Biosciences, (2) develop a simple field-based sample collection procedure compatible with the CRISPR detection cartridge (3) test the field deployable WSSV cartridge on the "PowerLite" prototype. This innovative technology would improve biomonitoring practices by enabling shrimp farmers to more rapidly and accurately screen for, and respond to, WSSV. It would provide a platform that could extend to other animal pathogens that impact US aquaculture and agriculture. This provides a unique business opportunity for Sherlock Biosciences to expand its technology platform beyond human health to domestic and global food security.
Project Methods
The reaction chemistry will be converted to a mix that has already been optimized by Sherlock Biosciences for compatibility. Because LAMP is common between the WSSV SHERLOCKv2 fluorescent assay and the Sherlock Biosciences optimized chemistries, the same WSSV target and LAMP primer set will be used and GMGI will share these materials with Sherlock Biosciences. The LAMP primer set targets WSSV viral protein 28. Because the SherlockTM optimized mix uses a different Cas enzyme than the WSSV SHERLOCKv2 assay (Ssp Cas12a instead of Aap Cas12b), a new guide RNA will be commercially synthesized to contain the scaffold sequence compatible with Ssp Cas12a. Sherlock Biosciences routinely uses guide RNAs synthesized by IDT in assay development.After obtaining the Ssp Cas12a guide RNA containing the VP28 spacer sequence, collateral cleavage efficiency will be confirmed in one-pot LAMP + Cas12a reactions in microwell format. Preliminary evaluation will be done using synthetic WSSV DNA target as input, and collateral cleavage reporter fluorescence will be measured with a qPCR machine. A robust reaction will yield a fluorescent signal ≥ 10,000X above background in under 20 minutes when one million copies of target is present. Next, the same evaluation will be done using genomic DNA as input isolated from a shrimp sample infected with ≥ one million WSSV copies previously quantified by the WSSV SHERLOCKv2 assay and qPCR. GMGI will provide Sherlock Biosciences with this input material. Then, a 10-fold dilution series of both synthetic and genomic targets will be tested to assess the sensitivity of the new chemistry. Assay conversion will be considered successful if the assay can detect ≤ 100 copies and can show distinguishable differences between dilutions (with fluorescence showing a Pearson's correlation of ≥ 0.8 with viral copies). The sensitivity will also be compared with that of the lab-based WSSV SHERLOCKv2 assay, and a Pearson's correlation above 0.5 for commonly tested sample dilutions will be considered successful. Should any of the above evaluations not result in success, reaction mix optimizations will be made to increase sensitivity and reduce time-to-result. This may include addition of a chemical additive (e.g. 200mM glycine), altering guide RNA:Cas12a ratio, altering dNTP and magnesium concentrations, temperature, the sequence length of fluorescent reporter, and the addition of displacement primers. Sherlock Biosciences has found all of these to improve assay performance. If sensitivity and time-to-result needs to be increased further, we will evaluate the benefits of multiplexing another LAMP primer set and guide RNA to target a different but equally conserved and specific WSSV genomic region. Finally, specificity will be evaluated by checking for cross-reactivity with clinically relevant titers of common viruses (n = 5 for each EMS, EHP, IHHNV, IMNV and TSV - infected shrimp) as well as estimating the false positive rate from specific pathogen-free shrimp (n = 20).After confirming sensitivity and specificity comparable to existing technologies, we will convert the assay from liquid-based microwell format to cartridge-based format by lyophilizing reaction components to be compatible with the PowerLite. The same reaction mix formulation and lyophilization procedure that led to success will be attempted. The reaction mix includes 10% trehalose and 1% dextran in addition to the same reaction components used in the liquid format above. If needed, enzyme levels can be increased to maintain reaction efficiency. Prior to lyophilization and to facilitate cartridge assembly, the reaction mix is added to liquid nitrogen to freeze all components and generate Lyo-beads. Lyo-beads will be tested for successful lyophilization through rehydration and microwell plate testing using positive and negative control purified shrimp DNA as described in Objective 1.1. A successful lyophilization will show a fluorescence signal ≥ 10,000X above background in ≤ 20 minutes for a shrimp sample infected with ≥ one million WSSV copies compared to a specific pathogen-free shrimp sample.Lab-based diagnostics require nucleic acid input to be purified, which has time and cost constraints. To circumvent these, different field-deployable sample collection methods will be tested for compatibility with the PowerLite device. Current human diagnostics developed for the PowerLite device process total nucleic acids from saliva or nasal swabs that are immersed in TE buffer pH 8.0 and heated at 90°C for 3 minutes in the presence of a reducing agent to inhibit RNase activity. We will attempt to use a similar nucleic acid preparation methodology on samples collected from either swabbing shrimp tissue or a lysate created through crude mechanical disruption. If needed, a small mesh screen can be added to the device to prevent the lysate slurry from clogging downstream components. We will first attempt this on abdominal muscle tissue of WSSV-infected shrimp. If the yield is too low, we will experiment with other tissue types (e.g. pleopods, gills) and lysis additives (e.g. 15 mM NaOH to help dissolve cell membranes).The effects of different sample collection methods on reaction efficiency will be compared initially using the lab based microwell fluorescent assay. Samples collected from highly infected shrimp will be compared to purified genomic DNA from infected shrimp (positive control, n = 20) and from specific pathogen-free shrimp (negative control, n = 20). A sample collection method that shows a positive agreement of ≥95% and a negative agreement of 100% will be considered a success. Next, the effect of sample collection on assay sensitivity will be tested by collecting samples from the same infected individual using different sample collection methods. Samples will be diluted in a 10-fold dilution series and tested in the lab based microwell fluorescent assay. Limit of detection will be compared across sample collection methods and the method that enables detection of ≤100 copies per reaction will be considered successful.Lyophilized reaction components created in Objective 1 will be assembled onto the PowerLite-compatible plastic cartridges manually and sealed with pressure-sensitive adhesive film in a dry box. The device uses a sample analysis algorithm to give a binary readout ('positive' or 'negative'). This device will be calibrated using purified genomic DNA isolated from a shrimp sample with high viral load (>1 million copies per microliter) and from a pathogen-free shrimp sample. These samples will serve as positive and negative controls to train the sample analysis algorithm to make appropriate calls. Once calibrated, we will test a dilution series to continue to train the device to distinguish between negative and positive samples at the limit of detection. After device calibration is complete and the device is consistently making correct calls, we will then test positive (n = 20) and negative (n = 20) shrimp samples prepared using the sample collection method determined in Objective 2. If there is less than 95% positive agreement and 100% negative agreement, or if the method negatively impacts the fluidic movement within the cartridge, the sample preparation method will be determined not suitable for the cartridge. Method modifications and alternative sample collection methods will be tested for compatibility with the cartridge until ≥95% positive and 100% negative agreement are reached. Next, on-device sample preparation will be tested using the determined sample collection method. A variety of infected individuals ranging in viral load (n = 30; previously quantified by qPCR and WSSV SHERLOCKv2 assay) will be sampled and run to assess sensitivity of the cartridge-based assay. In this initial diagnostic performance evaluation, a diagnostic sensitivity of ≥98% will be acceptable.