gene

drives

Read the assigned preprint titled: “Daisy Quorum Drives for the Genetic Restoration of Wild Populations”

In your own words, please answer the questions below:

1. What is the purpose of “Daisy Quorum” gene drive?

A CRISPR-based gene drive system is used to alter the genetic code of a population. The Daisy Quorum is a restorative safety system built into the DNA of the engineered organism, capable of reversing the gene drive at any time when needed. It can be seen as a detection mechanism to prevent catastrophic damage to ecosystems.

According to the Sculpting Evolution group, the purpose of "Daisy Quorum" is allow efficient, community-supported and genetically reversible ecological engineering. In other words, Daisy Quorum purposes a responsible use by allowing local communities to decide how to solve their own ecological problem.

“We need to get out of the ivory tower and have this discussion in the open, because ecological engineering will affect everyone living in the area” - Kevin Esvelt

2. What is your immediate emotional response after reading the preprint?

Overall, I get a positive feeling about this preprint. I appreciate the openness and the fact that full disclosure happens during the process. The Sculpting Evolution group at the MIT Media Lab takes a high personal risk for the purpose of opening up the discussion about if, when, how, where and with whom to implement a technology like this.

Students take the risk not to get a job elsewhere in academia.

The intended audience is academics, but this document is open to the public and they will read it. The paper is dense and without a background in synthetic biology, it might be hard to work through it.

Other questions that came to mind include:

The process of gene drives is horizontal and slow, so wouldn’t there be a more efficient way to solve the problem of ticks carrying Lyme’s disease?
Does the technology alter the behavior of the mice? For example, in the case of plastic nanoparticles that accumulate in the food chain and are known to alter the behavior of animals.

3. Describe a scenario, real or fictional (real-world is preferred), where you would deploy a technology such as “Daisy Quorum”.

The premise of a Daisy Quorum is to have a back-up plan to reverse the technology. The Daisy Quorum could be particularly useful in the context of mitigation strategies to combat climate change. For example, some geoengineering groups propose to release sulfate aerosols in the stratosphere, to temporarily lower the earth’s surface temperature. This technology aims to mimic the cooling effects that follow after a volcanic eruption. If this were to be a biological material, it would be great to have a Daisy Quorum to mitigate the negative effects when it turns out to fail.

Another example is ocean acidification, which is a reduction in the pH of the ocean over an extended period of time, caused primarily by uptake of carbon dioxide (CO2) from the atmosphere. This causes carbonate ions to be relatively less abundant. Certain fish's ability to detect predators is decreased in more acidic waters. This affects the entire food chain. A study can be deployed to find out if there are genes that can make a fish resistant to acidic waters.

https://oceanservice.noaa.gov/facts/acidification.html

The types of organisms suitable for gene drives are the ones that can reproduce. Gene drives is only effective when the reproduction cycle is fast: a maximum of two years is allotted for the technology to be worth the effort.

CLASS ASSIGNMENT

THEORETICAL HOMEWORK

KEVIN

ESVELT

LAB task

EXPERIMENTAL HOMEWORK

4. What would you be concerned about if a neighboring country, or city, decided to deploy “Daisy Quorum” technology?

I would be interested in the implications on the ecosystem outside its boundaries. What are the negative externalities of this technology? How long does it take for the edited genes to mutate? What are the ethical implications? In class, we have learned that genes mix between species, with the striking example of the fact that a contemporary cow has 25% snake DNA. What happens when the genes are shared with other species? Does the Daisy Quorum still work? How is it going to impact those species? Known predators of white-footed mice are weasels (Mustela), snakes (Serpentes), owls (Strigiformes), hawks (Accipitridae), falcons (Falconidae) and red foxes (Vulpes vulpes). These animals are higher up the food chain, how is the gene drive going to impact them?

On the website of Sculpting Evolution the following question is posed; 'Are there any risks posed by daisy drive systems?' and answered as follows: The major risk is that a rare event will move DNA encoding a drive component from one element to another, thereby creating a 'daisy necklace' capable of global drive. Since this kind of recombination depends on similarity of DNA sequences, we designed dozens of variants of the CRISPR components to come up with a set that aren't similar to one another, then worked with George Church's lab at Harvard to identify those that still work well enough to use. We should now have enough diverse components to build stable 6-element drive systems.

5. Is the name “Daisy Quorum” intuitive to you? If you were to name the technology, what would you name it? Note: we may actually adopt your name in the final publication!

Daisy reminds me of James Lovelock’s mathematical Daisyworld Gaia hypothesis. I had to look up the translation and meaning of the word quorum, which means the minimum number of members of an assembly or society that must be present at any of its meetings to make the proceedings of that meeting valid.

Other interpretations are:

The minimum number of members required for a group to officially conduct business and to cast votes, often but not necessarily a majority or supermajority.
A selected body of persons.

To me, quorum comes across professional and distant. It does indicate the exact technical function within gene drives, but might not ring a bell with people who aren’t familiar with it.

Keywords that could be used are: safety, security, safeguard, switch, back-up, reversible, community, biosafety net, fixable, restorable, prevalence, restoration, reversibio, transparent, erase-able.

Perhaps: Daisy erase me

You would want to reflect the collaborative nature of this research endeavor in the name of the technology. Essentially, the Daisy Quorum is a biological back-up system to reverse the gene drives. The technology is universally applicable to other species.

Additional feedback on the paper

Something that took a while to figure out when I was reading the paper for the first time was: what is the difference between the Daisy Quorum and gene drives? Further reading revealed that there are some significant differences. This might be a result of an overuse of the word ‘drive’. Daisy Quorum Drive and Gene Drive are both used and initially, it seemed to be interchangeable. I would suggest to leave out the word ‘drive’ in Daisy Quorum Drive, to differentiate better between the concepts and increase the readability of the paper.

Protocol

Make the following mixtures with the plasmids you were provided. Make sure that the total DNA concentration does not exceed 100ng/ul, and try to conserve materials. 10ul of plasmid mix is plenty for this experiment.

Transform bacteria with the EC-Rope plasmid (+- 1.5 hours)
1. Get crushed ice
2. Thaw
Plate bacteria on selective LB-Agar with 25 ug/mL of Carbenicillin
Place in 4 degree fridge the next morning
Make new competent cells that contain the EC-Rope plasmid
Make the following plasmid mixtures
- CRISPR-A: Ds-cas9 + PM-bla
- CRISPR-B: Ds-cas9 + PM-sp22a-!con
Transform two aliquots of competent cells with the two plasmid mixtures from step 4 (grow for an hour)
Plate cells on double selective LB-Agar plates containing Spectinomycin at 50 ug/mL, and Chloramphenicol at 35 ug/mL.
Pick out colonies and streak them out on LB-Agar with 25 ug/mL. Why did colonies carrying the CRISPR-B mix grow and the CRISPR-A mix fail? Hypothesis what happened. Feel free to use the plasmid maps provided as a guide.

Discussion

Colonies carrying the CRISPR-B mix should have grown because we inserted the antibiotic gene into the plasmid, whilst colonies with the CRISPR-A mix were supposed to be inhibited by the antibiotics present in the culture media.

Although these were the expected results, we ended up having colonies in both cultures. We believe something must have gone wrong during the mixing of reagents which could have led to growth on both plates.

The plasmid maps are displayed below.