Progress 08/01/16 to 12/31/17
Outputs
Target Audience:This is the final technical report and is intended for the use of the USDA. Changes/Problems:Reasons for slippage if established goals were not met Task 1 ran into a significant delay due to synthesis and production of the CRISPR/Cas multiplexed vectors. Production of these vectors by GeneCopoeia turned out to be a protracted effort taking months and, thereby, delaying the overall project. This delay was not anticipated, and we had already purchased PL shrimp for the WSSV oral challenge that, due to the delay, were oversized for the experiments and a new cycle of shrimp acquisition and growth was required. Due to these factors, BrioBiotech was allowed a no-cost extension by NIFA in order to complete the project. Task 2 milestones were not achieved (as described in more detail above). Our development of the primary cell culture methods was the primary stumbling block. Our company expert, Dr. Arun Dhar, left the company and the state. While he remained as an advisor, translation of these methods without his hands-on contribution to the project hampered its progression. We were able to produce live hemocytes that were viable for >7 days, but these did not become adherent cells that multiplied in our plates. We ran out of sufficient numbers of juvenile shrimp to generate viable but non-replicating hemocytes to the study and began growing additional animals from 0.2 g size PLs. About this time, the WSSV viral oral challenge studies showed unexpected impact of the WSSV CRISPR/Cas construct injection trial (Task 3) that reprioritized our efforts to validate (by a 2nd WSSV oral challenge trial) and to work up the data in the grant period left. What opportunities for training and professional development has the project provided?This project hired in a new graduate from Hood College and provided her with her first laboratory job. She got on the job training in molecular techniques and has moved on to a position at a much larger company focused on molecular services. She has kept in touch and is a star performer for that company and very happy with the training received at BrioBiotech. How have the results been disseminated to communities of interest?We are waiting for our NGS data to be analyzed and then intend to publish the results (if we demonstrated genome editing). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact No therapeutics are available to combat shrimp white spot syndrome disease (WSD) caused by white spot syndrome virus (WSSV). We explored whether genome editing can reduce WSSV infection or slow WSD progression. We designed vectors, ran two oral challenge studies, isolated DNA, ran qPCR, and formulated pDNA in MSP (Matrix/Scaffold/Payload) particles then tested release and stability. We analyzed for indels by amplicon size. Amplicon next generation sequencing was done; data not yet analyzed. Broader impact of this study will be that genome editing can reduce or delay WSSV infection and offers a potential WSD cure. Conclusions WSSV infection was slowed by injection of pDNA containing CRISPR/Cas9 elements. CRISPR/Cas vectors targeting 3 WSSV genes simultaneously were most effective. MSP particles carrying DNA released more DNA at pH 7 vs. pHs 2 & 5; delivery to hindgut rather than more acidic stomach, caeca and foregut. If nuclease-free MSP scaffold and fresh matrix materials used, pDNA successfully encapsulated and stable through production and for days at 4?C. Matrix components and high oleic sunflower oil (used for storage of MSP particles) protected pDNA if the materials were not oxidized. Additional research is required to demonstrate that WSD protection can be improved (better disease control) and to orally deliver an economically viable approach to WSD control. Task 1 Produce CRISPR/Cas constructs targeting essential WSSV genes 3 Milestones: 1) What are the best targets in the viral genome? (Completed). 2) Design and produce constructs (Completed). 3) Produce milligram quantities of DNA for further tasks (Completed) Target Selection: WSSV target genes: DNA polymerase, the major capsid protein VP28, and ribonucleotide reductase subunit 1 (DNApol, VP28 and RR1). Vector Design: Multiplexed vectors used; a single transfection event would hit 3 targets. Produced 3 vectors that each had 3 target sites (sgRNAs) and targeted a single gene (i.e., VP28, DNApol or RR1). These vectors should produce the endonuclease (Cas9) and crRNA/gRNAs targeting three sites on RR1, DNApol, or VP28. Task 2 - Test ability of CRISPR/Cas constructs to inhibit WSSV amplification in primary cell culture 2 Milestones: 1) Isolate and culture shrimp hemocytes (Not met). 2) Follow infection and show that the delivery of the CRISPR/Cas constructs inhibits in vitro (Not met). Designed sgRNAs and crRNAs and purchased Cas9 endonuclease. Isolated shrimp hemocytes that survived in microplates for days; they did not adhere and multiply. Therefore, obtained more shrimp to generate enough hemolymph to carry out our work with non-replicating but viable hemocytes. We ran out of time to complete Task 2. Task 3 - Test ability of CRISPR/Cas constructs to inhibit WSSV by injection 1 Milestone - Will WSSV infection and/or titer be reduced on challenge when first injected with constructs? (Completed). Initial WSSV oral challenge (GCRL #1) addressed the hypothesis that injection of CRISPR/Cas9 vectors targeting WSSV for genome editing would slow progression of WSD. Cumulative mortalities were monitored and began ~ 50 h post challenge, where there was a rapid rate of mortalities in the +Cont and large subunit of ribonucleotide reductase (RR1) tmt. Other tmts had reduced mortality rates and slowed progression of infection. No mortalities in -Cont. Four days post-challenge, RR1 and +Cont mortalities flattened out, while other tmts slowly reached equivalent cumulative mortalities after > 1 week post-challenge. GCRL#2 data generally mirrored GCRL#1. However, tmts targeting DNApol and VP28 showed rapid onset of mortalities unlike GCRL#1. While the mortalities in VP28 tmt flattened off, DNApol tmt mirrored +Cont, so no protective effect. The 3 vector and RR1 tmts both delayed WSD progression but slowly approached the same cumulative mortalities of the other tmts. No mortalities in -Cont. These results validated the trend of delay in progression of WSD but did not completely replicate GCRL#1, since the most effective tmt in GCRL#1 (DNApol) and least effective (RR1) traded places in GCRL#2 (most effective now becoming least effective). Cumulative mortality data generated showed a trend toward slowing the progression of WSD. The next step, if we demonstrate genome editing occurred, would be a dose response curve optimize the impact of genome editing. The presence of indels in target amplicons would be putative indication that genome editing had occurred. Survivors (S; animals alive at end of challenge study) and live-harvest animals (LH; animals that showed no signs of disease during the height of mortalities) had what appear to be deletion events in the target site amplicons. DNApol amplicons were found in all DNA samples (LH as well as S). Additionally, a band was present in the -Cont for DNApol primers; no indels were obvious and it is not clear why the -Cont had an amplicon of the predicted size. VP28, RR1_1 and RR1_2 primers did not have amplicons of the correct size in - Cont. VP28 had a low mol. wt. band that might be reactants rather than an amplicon. VP28 primers gave variable results in LH and S. RR1_1 primers gave amplicons of the correct size and some live had a smaller amplicon (~250 bp) that could be evidence of genome editing. RR1_2 gave at least weak amplicons in both LH and S. The same analysis was done on samples from GCRL#2. No indels were seen on the samples analyzed from GCRL#2. We are still analyzing the NGS data; if analysis shows genome editing occurred, we will publish these data on the potential for use of genome editing as a therapeutic for fighting infection by viral pathogens. Task 4 - Formulate CRISPR/Cas constructs in delivery vehicle for oral delivery in Phase II - evaluate stability and release 3 Milestones: 1) What are the best ways to stabilize and deliver DNA constructs? (Completed), 2) How stable are the constructs in optimal formulation to moisture and time? (Completed), 3) Are they released in appropriate conditions? (Completed) MSP particles containing DNA were tested at different pH's for DNA release. Data consistently show a rapid release of the DNA at pH 7 with little release at pHs 2 & 5. DNA release in synthetic intestinal juice increased over time while release of DNA in synthetic gastric juice was essentially static, maximum (and little) release immediately. Successful delivery of CRISPR/Cas constructs requires that the pDNA remain intact and functional during production of the delivery vehicle, incorporation into feed, storage and ingestion by the fish. MSP particle components had a negative impact on the pDNA. MSP particles containing a 2% protein payload were assayed to determine if they impacted the stability of the pDNA. While the supercoiled and nicked bands appeared to be fine, there was a low molecular weight nucleic acid component that may or may not be due to degradation by the particles (the protein payload may have some small nucleotides in it). When the pDNA was reacted with the water impermeable matrix materials that had been stored at room temperature for a month, some pDNA degradation was observed but no generation of small nucleic acids. High oleic sunflower oil, used as a carrier for the MSP particles, had no impact on pDNA. Testing the scaffold protein (SPI5) and a 10% SPI5 load in M13 particles showed that the small nucleic acids are not from SPI5. However, SPI5 reduces supercoiled band while increasing nicked band, indicating some nuclease activity. Two freshly made MSP particles with 2% loads of SPI5 showed no breakdown of pDNA. pDNA formulated in MSP Particles were produced with materials treated to remove nucleases. The pDNA survived MSP particle production and was intact after 4 days. There appears to be a small bit of breakdown DNA (at low molecular weight (~100 bp). We will have to do more research to see if we can prevent all damage on production. This small band does not decrease over time (at least 4 days).
Publications
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Progress 08/01/16 to 07/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Aim 2 - We have had trouble getting primary cell culture working in our laboratory. We can get the cells but they do not replicate as reported in the literature. We have discussed our issues with the authors of the primary papers on this to troubleshoot the problems and think we have potential solutions. Additionally, we have talked to experts in cell culture to see if they had suggestions. We have a number of potential fixes and are now waiting for the shrimp to grow to sufficient size for this study. What opportunities for training and professional development has the project provided?We hired a new graduate from Hood College for this project and provided her with training to complete portions of this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Aim 1 - completed Aim 2 - Shrimp are almost the size to be used for a renewed attempt to get the primary cell culture of hemocytes working. If this can be completed, the experiments are expected to be rapidly acheived. Aim 3 - We will complete processing the muscle tissue sampled in the first injection with oral challenge trial completed at GCRL and generate supporting data for viral load and genome editing. In addition, we plan to repeat the injection with oral challenge trial at GCRL to validate the promising results obtained in the first trial. If time permits, will process these samples as for trail 1 to get supporting viral load and genome editing data. Aim 4 - We will apply the CRISPR/Cas vectors produced in Aim 1 in the formulations that we have already tested with model nucleic acids and demonstrate stabilty during processing, storage, and model digestive juices.
Impacts
What was accomplished under these goals?
1) Produce CRISPR/Casconstructs targeting essentialWSSV genes Completed design and received delivery of three constructs that were designed in collaboration with Genecopiea to target specific white spot virus genes. Genecopiea used their proprietary software to design crRNAs that targeted three sites on each of three WSSV genes (DNApolymerase, VP28 structural protein and subunit 1 of ribonucleotide reductase gene). These were designed using the sequences from PubMed for the Chinese strain of WSSV. The Cas9 gene was codon optimized for expression in shrimp and cloned into each of these vectors. We designed each vector to have a shrimp promoter and T7 promoter in tandem in front of the Cas9(shrimp optimized) and the codons for multiplexed expression of the sgRNAs (three for each vector targeting the same gene). These vectors were amplified to produce sufficient DNA for use in the injection trials in Aim 3. 2) Test ability of CRISPR/Casconstructs to inhibit WSSVamplification in primary cellculture We have run into difficulty getting primary cell culture of shrimp hemocytes to work in the lab. This work continues but was hampered by a lack of shrimp. We are growing more shrimp and should return to this aim shortly. All synthesized materials (sgRNAs) are ready for this work and we just need to get the primary cell culture to work. 3) Test ability of CRISPR/Casconstructs to inhibit WSSVby injection The first run of the injection studies have just been completed at the Gulf Coast Research Laboratory in collaboration with Dr. Steve Curren. The trial showed that all of the treatments (CRISPR multiplexed vectors designed and amplified in Aim 1) provided some delay in the onset of morbidity/mortalities in the shrimp compared to the positive control (mock injection). The protection from the vector expressing subunit 1 of the ribonuleotide reductase was less than the other vectors with mortalities catching up to the positive control in about 80 hours post challenge with WSSV. While complete control of the disease was not achived with the other constructs, considerable delay in the progression of the infection was observed for the vectors expressing VP28 and DNA polymerase targeted sgRNAs. At least for the first run, DNA polymerase targeted vectors provided the best protection from WSSV infection. A combination of all three vectors (each at 1/3rd the strength) and VP28 targeted sgRNAs provided significant delay in the progress of the disease but less than seen with DNA polymerase. We are currently extracting the DNA for viral load determinations and collected samples to verify the effect of the disease via histology. We will be repeating the oral challenge study to verify the mobidity/mortality results before the end of the Phase I project. 4) Formulate CRISPR/Casconstructs in delivery vehiclefor oral delivery in Phase II -evaluate stability andrelease We have tested several different formulations of the MSP system using a protein scaffold using ribonucleotides and salmon sperm DNA as models for the vectors. A specific formulation has been developed that seems to hold promise of protecting the DNA during low pH and processing but quickly releases the DNA at neutral pH (similar to that seen in the intestines). This will be applied to the vectors in Aim 1 shortly.
Publications
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