Team:Lethbridge/RNAi

Lethbridge iGEM

• Project
• Description
• Design
• Experiment
• Database
• Notebook
• Parts
• Safety
• RNAi
• Practices
• Integrated Practices
• Contact
• Interviews
• Literature Review
• Ridealongs
• Protocol
• Sampling
• Results
• Microbiome
• Antibodies
• RNAiCare
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• Attribution
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menu

• • Project
    • Description
    • Design
    • Experiments
    • Database
    • Notebook
    • Parts
    • Safety
    • RNAi
• • Practices
    • Integrated Practices
    • Intro
    • Interviews
    • Literature Review
    • Ridealongs
    • Protocol
    • Sampling
• • Results
    • Microbiome
    • Antibodies
    • RNAiCare
• • Team
    • Members
    • Collaborations
    • Attribution
    • Sponsors
• Achievements

RNAi

Background

Our strategy for dsRNA production is a multi-part approach. The construct is expressed dicistronically, with a His-tagged MS2 coat-protein expressed initially. Additionally, we employ the use of a Herpes Delta Virus Ribozyme (HDVR), which will be evolved through SELEX in order to generate a thermozyme (Win, M.N., Smolke C.D. 2007). Upon an increase in temperature (22 °C to 37°C), the thermozyme undergoes a conformational change, resulting in cleavage at a specifically prescribed nucleotide. This thermozyme is placed just upstream of an MS2 coat-protein binding site. This allows us to purify only full-length RNA once it has been transcribed, as those transcripts not harbouring the binding domain will not be effectively purified. His-tagged MS2 coat-protein is then free to bind the newly transcribed binding domain. Using affinity chromatography, purification of the MS2 coat-protein and the bound RNA is possible. Upon a temperature increase, cleavage and liberation of a single stranded RNA occurs, allowing for purification of a single strand of highly pure RNA.

By using two complementary RNA-generating sequences within the thermozyme construct, we are able to generate double stranded RNA for use in pest control simply by annealing the two resultant strands produced by our purification strategy.

Given our chassis and ability to over-express His-tagged MS2 coat-protein, our purification strategy is poised to purify large amounts of highly specific RNA. The scalability of this platform lies in the ability of individuals to design novel pesticides for any target organism, having only requisite knowledge of the genome. New dsRNA-based pesticides will be employed cheaply and specifically without costly design and massive amounts of resources currently utilized in the development of novel pesticides. Pesticides represent a multi-billion dollar industry worldwide, and with the scalability of this synthetic biology mode of production, this project represents a readily commercializable method of producing large quantities of highly specific pesticides applicable to a wide array of pest species.

For large scale production, our group will likely use fermenters for sufficient growth. However, as demonstrated, the concentrations of dsRNA required for gene silencing are sufficiently low that the large-scale production need not necessitate massive amounts of resources.

Human Practices

As a part of the design of our project, we looked over the ethical implications of our study. We looked into a variety of different farming methods and uses of pesticides. We examined off-target and non-target effects by ensuring the target sequences we selected were compared against the entire database of sequenced genomes using the NCBI BLAST program. We also spoke with various experts and held a panel discussion regarding the efficacy of our project from the lab to the field.

We also contacted major small molecule pesticide distributors to estimate the current cost analysis. Finally, we collaborated with the Canadian Food Inspection Agency (CFIA) and Health Canada to further explore the ethical implications of our project.

Risks

RNAi: Off-Target and Non-Target Effects

The RNAi is used more extensively in pesticides due to its target specificity. However, it does not come without the possibility of negative effects or errors in what RNAi sequences are targeted. Ideally, the RNAi manufactured should only target the specific sequence that causes virulence in Fusarium graminearum which would be an on-target effect. Due to sequence similarities across species and within the genome of the target pest, there could be unwanted effects. Two types of these effects are off-target and non-target.

Off-target effects are when the processed RNAi silences a part of the genome that is downstream or upstream of the target sequence, or only part of the target sequence. RNAi technologies have been found to have a lack of sequence specificity [1]. Therefore, a virulence factor may not be targeted, allowing F. graminearum to continue infecting crops. A 100% sequence match is often required for the RNAi to work [2], any slight sequence change or mutation could render the RNAi sequence unable to disrupt the target sequence. Therefore, important to know what the exact sequences are that will cause death in the organism when down-regulated. Just because a 100% match is required for the RNAi to work doesn’t mean that it can’t interact with and affect another area within the genome that is slightly different, impairing its function as well. Further testing of the RNAi spray would be required to determine the exact effects regarding the probability of off-target effects, but it has been shown that using a lower concentration of interfering RNAs reduces the amount of off-target effects within the genome [3].

Non-target effects are when the RNAi down-regulates a gene that is not in the intended target species. Due to the short length of interfering RNAs, there is more opportunity for sequence similarities between species. Therefore two different species could be equally likely targets of the RNAi. This means that the spray could influence the growth and function of other pests, pollinators, and crops and even cause death in these organisms. Similar to off-target effects, it is possible that due to lack of specificity a non-target species could have a sequence with a completely different function than the target which interacts with the RNAi. It is important to determine what species in agricultural communities could be affected by the chosen sequence, and what sequence similarities exist between species that are closely related to the target.

Off-target and non-target effects could potentially occur at the same time. For example, a non-target species could have short lengths of sequence similarity with F. graminearum and have its genome disrupted by the RNAi, but due to RNAi’s lack of specificity there could be off-target silencing downstream of the matching sequence within that organism’s genome.

Sequence BLAST

To examine potential off-target effects of our gene-silencing dsRNAs, we undertook an extensive comparative approach using NCBI BLAST. Areas of similarity within other genomes may result in potential silencing outside of the desired organism. We first compared our chosen sequences to the entire database of sequenced genomes. After finding minimal overlap between our sequences and any other off-target organisms, we moved on to compare specifically within humans and varietals of wheat. Again, we found no significant overlaps or risks in terms of silencing capability. Therefore, we are confident in the safety and specificity of our design platform.

Our search for potential off-target effects represents an exemplary level of attention to the safety of consumers as well as those handling and producing the dsRNA-based pesticides.

We tested our sequences against common species.

References

Jackson A. and P. Linsley. (2010). Recognizing and avoiding siRNA off-target effects for target identification and therapeutic use. Nature, 9, 57-67.

Subba Reddy Palli. (2014). RNA interference in Colorado potato beetle: steps toward development of dsRNA as a commercial insecticide. COIS, 3, 1-8.

Dharmacon. (2014). Off-Target Effects: Disturbing the Silence of RNA interference (RNAi). 1-4. 


Stakeholders

RNAi: Off-Target and Non-Target Effects

Wheat crops in Canada are protected from infection by Fusarium graminearum through the application of foliar fungicides and seed treatments. Foliar fungicides are sprayed onto the plant itself rather than soaking seeds as is done in a seed treatment. Seed treatments are used to prevent initial FHB infections from the dormant chlamydospores in the soil. These seed treatments are however, not as effective in prevention of the spread of FHB once the crop begins to grow and are most vulnerable [1]. A suppressive spraying method is used, meaning crops are sprayed at the point in growth that they are most vulnerable to infection by FHB. Farmers will spray once after 75% of the heads on the main stem are fully emerged, but before 50% of them have flowered [2].

Table 1: Product information regarding foliar fungicides that target FHB in winter wheat [3][4].

Fungicide	Recommended Vol. / Acre (mL)	Cost/Acre	Concentration (g/L)
Folicur EW	201	$12.13	250
Prosaro	324	$19.62	250
Fuse	118	$11.67	432

Table 1 provides information about three commonly used FHB combatant foliar fungicides. The cost of spraying these products adds up when considering the millions of acres that Canadian farmers must spray to keep their flowering wheat Fusarium-free. Seed treatments can also be quite expensive. For example, a fungicidal seed treatment made for wheat called Vibrance Quattro is $346 per bushel at 69.0g/L [4]. These costs all may vary based on the retailer and composition of the fungicides. Our goal is to eventually create a way to produce RNAi that is more affordable than these conventional fungicides.

References

[1] Agriculture and Agri-Food Canada. 2010. Crop Profile for Winter Wheat in Canada, 2010. Pesticide Risk Reduction Program Pest Management Centre Agriculture and Agri-Food Canada. 1-65.

[2] Government of Saskatchewan. 2015. Fusarium Head Blight. Provincial Crop Protection Laboratory. http://www.agriculture.gov.sk.ca/fusarium-head-blight

[3] Bayer CropScience Canada. 2015. http://www.cropscience.bayer.ca/Products/Fungicides

[4] Syngenta Canada. 2015. http://www.syngentafarm.ca/Productsdetail/Fuse/

Wet Lab

In order to generate a Herpes Delta Virus thermozyme1, we first constructed a library of variants to be utilized in systematic evolution of ligands by exponential enrichment (SELEX). To generate our library we incorporated random nucleotides into the U1A-RBD stem loop with either N9, N10, N11 or N12 random nucleotides for a total of 1.7 X 108 potential combinations. All of these variants were then assembled into the HDVR through oligo assembly followed by overlap extension PCR. Upstream of the HDVR there is a MS2 binding domain and a T7 promoter with an XbaI cut site at the 3’ end and a SpeI cut site at the 5’ end. A second module was used in order to generate an MS2 variant that will no longer dimerize and assembly into a viral capsid (BBa_K2109108). This was accomplished by using a characterized MS2 variant (V291-d1FG)2 with an N-terminal HIS tag, controlled through a T7 promoter and strong RBS followed by a double terminator (B0015). The MS2 will be overexpressed and used to saturate a Nickel Sepharose column so that the Thermozyme library can be applied to select for positive mutants. Two HDVR controls were generated (BBa_K2109109, BBa_K2109110) the first is a U1A mutant which was used as a negative control. The second HDVR mutant U1A-C75T is a negative control that can be induced to cleave in the presence of imidazole1. te (10 ng per PCR in a 50 uL volume) Clone into pJET and/or pSB1C3 for submission to iGEM parts repository.

References:

1. Ke, A., Zhou, K., Ding, F., Jamie, H.D., Doudna, J.A. (2004). A Conformational Switch Controls Hepatitis Delta Virus Ribozyme Catalysis. RNA, 13: 1384-1389.

2. Batey, R.T., Kieft, J.S. (2007). Improved Native Affinity Purification of RNA. RNA, 429: 201-205.