Evolution, CRISPR Gene Drives, and Ecological Engineering

4 November – videos (review10 on vimeo, lesson11 on vimeo) – Kevin Esvelt (Wyss Institute)

“Natural selection… is a power incessantly ready for action, and is as immeasurably superior to man's feeble efforts as the works of Nature are to those of Art.” - Charles Darwin

Evolution is the central paradigm of biology, but one of the least appreciated in bioengineering. Electrical circuits do not evolve; genetic circuits do - and usually break. Human-designed technologies minimize interactions to enable modularity; genetic circuits evolved to promote a complex web of interactions that inhibit modular design. These factors cause problems for bioengineers in the laboratory, but even more so in the wild.

Because wild organisms have been selected for efficient reproduction in their ancestral habitat, altering them almost always decreases reproduction. Hence, releasing engineered or selectively bred organisms into the wild has little if any lasting impact because natural selection weeds out the human-made changes.

Not all genes are so thoroughly tied to their host organism. Gene drive occurs when a DNA sequence ensures that is inherited more often than normal. Any sequence that causes gene drive can spread itself - and nearby DNA sequences - through populations even if their collective impact makes each organism less likely to reproduce.

We will discuss CRISPR genome editing, how it enables us to build gene drives, and questions of whether, when, and how we should develop synthetic gene drive systems to address real-world ecological problems.

Popular science overview of CRISPR gene drives: Esvelt KM, Church GM, Lunshof J. (2014) Gene drives and CRISPR could revolutionize ecosystem management. Scientific American (blog).

Technical discussion of CRISPR gene drives (skim as needed): Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014) Concerning RNA-guided gene drives for the alteration of wild populations. eLife, doi:10.7554/eLife.03401

Optional resources compendium

What features would you want to see in an online discussion platform devoted to guiding the development of gene drives? Assume that the researchers involved in the project are interested in soliciting public feedback before and during experiments so that they can better identify problems and redesign the technology. Please give specific examples of already-existing elements – if you want a discussion forum, should it be more like reddit, Quora, or something else? What should moderation be like?

What would it take to get you to regularly participate in such a community?

Identify a problem that could be addressed using a CRISPR gene drive.

Which organism would you target and how would you alter it?

Why is a gene drive a good solution relative to other options?

What could go wrong? Don't go into detail, but list several possibilities.

Who should be involved in the discussion of whether to consider this application?

Design a basic but evolutionarily stable gene drive that should function in your organism.

(If you find this challenging, see the proposal to build a gene drive capable of eradicating schistosomiasis.

Your goal is to design a proof-of-principle experiment that will determine whether CRISPR gene drives can function efficiently in the target organism. Your drive system should not cause population suppression, carry any 'cargo' genes, or change the sequence of any protein produced by the organism. It should only spread itself.

If the genome sequence for your organism isn't available or is hard to work with in any way, use the nematode worm Caenorhabditis elegans instead – we may use your design for a hands-on experiment in next year's class.

1) Identify a gene that is important for fitness in your target organism. Why must gene drives target genes important for fitness? A literature search may be required. You can use NCBI for information on genes if you wish, though Wormbase is generally superior and easier to use.

2) Find CRISPR target sites in the 3' end of the gene using an online tool. For C. elegans and many other organisms, a good user-friendly tool is GT-Scan. Recommended parameters: set the high-specificity mismatch limit to 3 and leave all other settings at default values.

More exotic species require less user-friendly software. For bonus points, use sgRNAcas9, which allows you to analyze any downloaded genome sequence in fasta format. Warning: it's not particularly user-friendly.

List the relevant criteria for inclusion of each target sequence (e.g. potential off-targets etc) as provided by the program you used.

3) Would it be possible for you to safely build this gene drive in your current laboratory? Which confinement strategies and safeguards would you use? See Akbari et al. and especially here for recommended confinement strategies.

Extra credit:

4) Generate a version of the target gene that will be carried with your gene drive. Recode the target gene so that it encodes the same protein, but does not have the sequences you are targeting. Should you change only those codons, most, or all of them?