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Gene Drive Systems in Nematode Worms

Developing methods of using nematodes to study and to predict the evolution of gene drive systems

Organisms: the nematode worms C. elegans and C. brenneri

Laboratories: Esvelt (MIT)

C. elegans:
Always employed: Ecological confinement (not found in New England), split drive (separated drive components), wild populations seldom reproduce sexually (resistant to drive).
Sometimes employed: Synthetic site targeting (drives cannot cut wild-type sequences)

C. brenneri:
Always employed: Ecological confinement (exclusively tropical), split drive.
Sometimes employed: Synthetic site targeting (drives cannot cut wild-type sequences)

Rationale: We have little experience working with systems that will evolve over many generations, which makes it difficult for us to predict the likely behavior of gene drive systems in the wild. Any deliberate release would be recklessly premature without some way to experimentally studying outcomes in the laboratory.

Key questions to answer include:
- To what extent will drive systems remain stable as opposed to changing over generations?
- How likely are they to spread into closely related species?
- Will these 'cyclic drive' systems to maintain costly traits behave as anticipated over multiple replacement cycles?
- To what extent and how quickly can immunizing reversal drives undo changes spread by earlier gene drive systems?

The problem is that none of these questions can be answered by studying gene drive systems in the species to be altered. Wild populations are so much larger than laboratory colonies of the same organism that traditional laboratory studies will not be sensitive enough to detect rare but critical evolutionary events that could occur in nature.

We need an organism that can be readily grown in vast numbers in the laboratory and reproduces quickly enough to obtain answers in a short period of time.

Experimental Plan:
Microscopic nematode worms are unique in that we can grow billions of them in the laboratory – and they reproduce so fast that fully 100 generations occur within a year's time. They are consequently the only model organism that can approximate the evolutionary dynamics of gene drive elements in the wild. The nematode genome can be easily engineered to encode the exact sequences of proposed gene drive elements, their target sequences, and those of related species. This will let us study particular drive systems in a variety of fitness and gene flow contexts.

To visually determine whether a given worm encodes a particular drive system, we will encode fluorescent markers in the genome to be erased or copied as the drive systems spread. We can pour worms from a liquid culture onto a plate, take a picture using a fluorescent microscope, and use existing software - a program called ImageJ with an extension that automatically identifies worms - to determine exactly how many worms of each type are present.

We are currently working with the model nematode worm C. elegans, which is not found in the New England area. This species normally consists of self-fertilizing hermaphrodites, which are highly resistant to gene drive systems because they seldom mate with one another. We're working with a particular laboratory strain with a mutation in the fog-2 gene that can only reproduce sexually. In parallel, we're experimenting with C. brenneri, an exclusively tropical species that naturally reproduces sexually.

Our work will provide vital information on the effectiveness of safeguards for controlling the spread of gene drive elements and dramatically improve our ability to predict their behavior upon release into the wild.

Nematodes silence foreign genes in the germline. We are currently licensing CRISPR proteins in these species by adapting methods pioneered by Craig Mello's group. Simultaneously, we are constructing split-drive systems that will function in licensed strains that express Cas9. Each drive system or its target site is labeled with a fluorescent protein, which will enable us to track the relative frequency of drive systems by simply measuring fluorescence at different wavelengths. To accelerate these efforts, we are building and optimizing a computer-assisted microinjection apparatus for high-throughput worm transgenes.