Team:Queens Canada/Side/KillSwitch

By inserting genes into C. elegans that enable it to identify and degrade harmful environmental toxins, we also risk making the engineered organism better suited for its environment than the native C. elegans strains. The modified strain may become invasive due to its enhanced chemotaxis and digestive abilities that may make it better at finding and digesting different foods or evading dangers. In essence, they have the potential to act as invasive alien species to the ecosystem, and once established, they have the potential to transfer to other ecosystems as well. Since our chemotaxis system is designed to target pollutants, it may also target other bacteria responsible for degrading pollutants and have an overall negative effect on remediation. In addition, human health concerns are another issue.

The creation of a novel-phenotype species comes with unknown risks about their pathogenesis. While it is unlikely for C. elegans to cause human pathogenesis (soil dwelling, nonparasitic nematode), it is not entirely out of the question, as other nematodes (such as those of the superfamily Filarioidea) are classified as parasitic, using humans as their host and causing disease.

Thus, to prevent these and other potential issues, we seek to design a genetic hinderance or “kill-switch” that will prevent the propagation of our modified worm. In C. elegans, several types of kill switch have been proposed, however only two of these methods will be expanded upon:

1. the MRT-2 mutant Kill Switch
2. the RNAi Kill Switch

Mechanism Overview

The triggered death of an engineered organism cannot always be assured using gene switches, as one simple evolution or mutation may render the mechanism ineffectual. A proposed control for our organism is an indirect method of kill switch where, instead of initiating the death of each individual engineered worm, the worm’s lineage and therefore, the genetic advantages given to the strain are limited to a certain number of generations. Through the insertion of our construct into a mutant, mortal germline of C.elegans, our transgenic worm lineage has a “date of expiration” where the genetic advantages given to it cannot be passed on further than a certain generation.

C. elegans’ normal germline is immortal, meaning that the genetic information within a worm’s lineage can be passed through generations indefinitely. However, mutants of this species can be found where the germline is mortal. In such mutants, after several generations of healthy specimens, sterile worms are finally produced from all predecessors. This then ends the lineage of the mutant strain of worms. Upon the death of the final generation (and death of all previous generations) the genetic information constituting the worm lineage will die and become extinct. A study done in 2000 has found continued telomere shortening and accumulation of end to end chromosome fusions in all later generations of the C.elegans mutant strain mrt-2 [1]. Telomeres are repeating DNA sequences that cap the ends of linear chromosome, preventing them from being degraded during chromosome replication or from fusing with neighbouring chromosomes [3]. The mutant strain was also found to be highly sensitive to DNA damaging agents and was shown to have chromosome loss at all times. This eventual complete eradication of telomere caps from linear chromosomes and hence chromosome circularization perhaps leads to the sterility of the final generation [2].

This study inspired us to create an indirect kill switch (as mentioned above) using a mutant worm lacking the gene mrt-2, one of the many genes involved in germline immortality. All of our engineered worms will be mrt-2 mutants and into them we will inject our genetic construct. Given that the germ-line of the mrt-2 mutant has a limited number of generations in which it can reproduce, our created strain is timed for eventual extinction of a span between 2 months and 1 year, with the average being 6.25 months.

Advantages & Disadvantages

This method of control has several benefits. The purpose of our project is to create a vehicle which can be used easily in soil to detoxify the soil’s pollutants. A switch which acts directly on a singular worm to kill it provides a small window of time in which to perform the bioremediation process intended. As toxic spills may vary in size and concentration, this time frame is less than optimal. With the mrt-2 method, our transgenic worms can multiply, passing the given traits through to their progeny until the designated final generation is reached. This last generation is infertile, resulting in the end of the transgenic worm lineage with the death of this last generation (assuming the death of all worms in previous generations). This solution would provide a much larger window of opportunity for the worms to continue the bioremediation process. With an average worm lifespan being between 2-3 weeks and mrt-2 mutants reproducing for 4-16 generations (median 10), the maximum time available for the worms to carryout the process is approximately one year (given maximum generations and maximum life span) with the average being 6.25 months and the minimum being 2 months [1].

A clear weakness to this method is that the generations of worms before the last are perfectly healthy and available to mate with other worms which are not mrt-2 mutants. This provides a problem, as the lineage of the mrt-2 mutants which contains our genetic constructs could be passed on to and carried through an immortal germline. This method is clearly imperfect, however, it should be noted that the hermaphroditic worms tend to self fertilize and only one in 500 worms are male, making this a viable (although untested and incomplete) idea.

The Knockout Pathway

RNAi knockout is a method of gene regulation where dsRNA is introduced to the worm, binding to gene products such as specific RNAs (mRNA), thereby decreasing or eradicating RNA activity by recycling said products. The RNAi pathway is initiated by the enzyme Dicer which cleaves long dsRNA (double stranded RNA) molecules into short fragments of about 20 nucleotides that are called siRNA (small Interfering RNA). These molecules of siRNA intereact with the RNA-induced silencing complex (RISC) which seeks out strands of RNA complementary to the siRNA and leads to the degredation of the target RNA.

Within the worm, exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans which activates Dicer. RNAi is remarkably potent (i.e., only a few dsRNA molecules per cell are required to produce effective interference.

Perfecting the Switch

Lastly, it is important to choose a gene to target that will sterilize the worm indefinitely. We have considered targeting one or several genes from the reproductive pathway. Some of these include spe-9, spe-11, spe-38 or mre-11. An example of a gene that would result in death when knocked out would be cdk-1 which is required for cell progression through the M phase in mitosis and meiosis.

In order the maximize the effect of the RNAi treatment, it is possible to select specific strains that show increased sensitivity to RNAi. A study conducted by Simmer et al. (2002) demonstrated a strain of C.elegans that is hypersensitive to RNAi treatment [8]. The mutant strain, rrf-3, which is the result of a loss of function of a putative RNA-directed RNA polymerase, showed more phenotypes for a set number of dsRNAs.

It is important for the kill switch to be as effective as possible. If our kill switch was leaky it could potentially result in our modified worm running rampant and destabilizing the ecology of the environment in which it has been introduced. In order to maximize the effectiveness of this kill switch, we must take into consideration the susceptibility of the worm to RNAi, whether the stage of life of the worm will affect its susceptibility and whether or not the effect of RNAi will be transferred between generations.

1. Ahmed S, Hodgkin J. (2000) MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature, 403:159-164
2. Smelick C, Ahmed S. (2005) Achieving immortality in the C. elegans germline. Ageing Research Reviews, 4(1):67-82
3. Telomere Shortening and Damage (2009) (http://www.immortalhumans.com/telomere-shortening-and-damage/)
4. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391(6669):806-11
5. Tabara H, Grishok A, Mello CC. (1998) RNAi in C. elegans: soaking in the genome sequence. Science, 282(5388):430-1
6. Timmons L, Fire A. (1998) Specific interference by ingested dsRNA. Nature, 395(6705):854
7. Timmons L. (2006) Construction of Plasmids for RNA Interference and In Vitro Transcription of Double-Stranded RNA. Methods Mol Biol. 351:109-17.
8. Simmer F, Tijsterman M, Parrish S, Koushika SP, Nonet ML, Fire A, Ahringer J, Plasterk RHA. (2002) Loss of the Putative RNA-Directed RNA Polymerase RRF-3 Makes C. elegans Hypersensitive to RNAi. Current Biology, 12(15): 1317-1319.