Team:Lethbridge/Practices Risks

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Practices

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

1. Jackson A. and P. Linsley. (2010). Recognizing and avoiding siRNA off-target effects for target identification and therapeutic use. Nature, 9, 57-67.
2. Subba Reddy Palli. (2014). RNA interference in Colorado potato beetle: steps toward development of dsRNA as a commercial insecticide. COIS, 3, 1-8.
3. Dharmacon. (2014). Off-Target Effects: Disturbing the Silence of RNA interference (RNAi). 1-4.