Propagating mutations throughout an entire population

Major strides in genetics from Gregor Mendel to Barbara McClintock have changed the way we see how genes are inherited. Because of this, we can calculate with reasonable confidence how genes will propagate from one generation to the next. But what if a scientist wants to bias how some alleles are transmitted, increasing their chance of being spread to the next generation? This can already happen in the naturalworld. Transposable elements, for example, can insert and remove themselves throughout the genome. In a recent article, scientists developed an ingenious technique to create homozygous mutations that pass to the next generation. This method can completely transform the genome of an entire population after several generations. Using the CRISPR-Cas9 system, Scientists from UC- San Diego created a mutagenic chain reaction (MCR) to greatly alter how genes are inherited.

To start, the researchers developed a bacterial construct that contained a Cas9 endonuclease and a gRNA that targets a gene of interest surrounded by homology arms that match sequences in the gene of interest. They inject this construct along with already made Cas9s and gRNAs to cut one allele of the gene and allow the construct to integrate using homologous recombination. The inserted construct will be transcribed, creating the Cas9 and gRNA which will then make a cut on the other chromosome. This allows for another round of homologous recombination, and the knockout of the other allele of the gene. This will interrupt the gene of interest, likely rendering it silent. Not only will insertion help create double knockouts, but when passed on to the next generation, the normally heterozygous mutations will now knockout the allele inherited from the other parent. This will create homozygous knockout progeny.

Their work show that after incorporating the cassette as an embryo, flies would emerge as homozygous knockouts. Even more incredible, the next generation showed complete knockouts. The gRNA targeted a gene in the X chromosome, that when homozygously mutated produced a lighter, yellow tinted fly. If the cassette was injected into a male fly, knocking out the gene, and this fly then mated with a wild type female, they found 100% of the female progeny were homozygous mutants, yellow, and all the males were wild type, a normal color. If the cassette was injected into a female fly, and mated to a wild type male fly, the investigators found 97% of the progeny were homozygous knockouts. In normal Mendelian crosses, one would expect 0% of the progeny to be homozygous for the knockout.

One name for such a system is a gene drive. The idea has been around since the 1960s and has picked up steam in the past years. Until recently, it had remained theoretical. However, with the major advances in genome engineering, it has now become a reality. We have seen similar work to what is presented here done in yeast at George Church's lab at Harvard. This technique has a lot of pros. It drastically decreases the amount of time it takes to make a stable mutant line. Also, there is less risk losing the mutation through mishaps with breeding (although this could be seen as a con if one fly escapes into another mutant line). Doing genetic screens too will be far easier and faster with this technique. A key use for this system is delivering transgenes into pests or disease carrying insects like mosquitos to help eradicate the spread of deadly diseases. In a similar vein, gene drives could help control invasive species. There are, however, some severe downsides to this method.

The most obvious downside to willingly releasing an organism into the wild with a gene drive are unseen results of the mutation. Nearly permanent changes introduced into a species will likely have many unknown effects on the population and environment. Furthermore, other mutations could occur through chance or off-target effects creating unforeseen mutants. Releasing these creatures into the wild in the hopes it will change the species for the better is incredibly hazardous. The risk of working with this protocol in the lab has drawbacks as well. If one is working with animals like flies or mice (although, to my knowledge, this hasn't been tested in mice), they can escape labs and potentially spread the homozygous mutations to the wild populations. There is a chance that the induced mutation would decrease the fitness of the animals, eventually weeding itself out of the population, but that isn't something we can depend on.

The authors acknowledged that there are substantial risks involved with such an experiment. They went through extensive steps to prevent the escape of animals. To George Church, however, it's the escape of the protocol that is dangerous. My thoughts are that with enough regulation, research with these methods can be done safely, but it must be taken seriously. There are several measures that would limit the broad impact of the experiments. One could target specific genes only found in a small subset of the greater population. Propagation could also be made in a way that it is easily reversible. In the work linked to above using gene drives in yeast, the authors split the locations of the Cas9 and gRNA, preventing the organism from being completely sufficient in driving changes throughout the population. The broader impacts of gene drives are enormous and we must take strides early by starting a dialogue and holding regulatory meetings to prevent any catastrophe.

Gene drives present an ethical conundrum. There is a thin line between positive results, and potentially dangerous mutations running rampant. It is imperative that measures are put in place quickly to contain all aspects of the materials and limit outside exposure. The invention and use of such a superb technique needs to be used safely and with great care.

References:

Bohannon, J. Biologists devise invasion plan for mutations. Science, 347, 1300 (2015).

Burt, A. Site-Specific selfish genes as tool for the control and genetic engineering of natural populations. Proceedings of the biological sciences B, 270, 921-928 (2003).

Esvelt, K.M., Smidler, A.L., Catteruccia, F., Church, G.M. Concerning RNA-guided gene drives for the alteration of wild populations. eLife, 2014;3:e03401.

Gantz, V.M., Bier, E. The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science Express, DOI: 10.1126/science.aaa5945 (2015).

Image credits:

Both images were made by the author of this post, with inspiration from figures in Gantz & Bier 2015.