Most people reading this blog today will probably consider this slightly old-fashioned, but I received a newspaper clipping from my mother in the post yesterday (yes, I mean a physical piece of paper that arrived in an envelope with a postage stamp on it). The title of the article (online here), which was published in the Austrian quality daily newspaper “Die Presse”, roughly translated to, “Genetic engineering is spiralling out of control”.
The article was referring to an experiment conducted by Gantz & Bier (2015) in which they invented a “mutagenic chain reaction” (MCR) based on the CRISPR/Cas9 technology in the fruit fly Drosophila melanogaster. Their experimental rationale is illustrated in this figure, which I copied directly from the paper:
The idea was to create homozygous insertional deletion mutations of a target gene, in their case the yellow gene in Drosophila. When yellow is mutated heterozygously in females (i.e. in only of the two genomic copies) the flies look normal/wild-type because mutations in this gene are recessive and the gene is located on the X chromosome; only once both copies are mutated does the phenotype manifest: albino flies. Since males only have a single X chromosome they are always albinos if they inherit the mutated yellow gene. Gantz and Bier built a DNA construct that expresses Cas9, the DNA endonuclease, and the guide RNA targeting yellow in both somatic and germ-line cells. However, the special feature of their construct was the flanking homology arms (HA; depicted in red): these facilitate the insertion of the construct at the site of cleavage by Cas9 via homology-directed repair. This means that the genome now stably expresses one copy of the Cas9/gRNA combination. Next, the second copy/allele will be targeted and again the construct is inserted in the DNA, disrupting the yellow gene. If this approach didn’t work you would expect first generation female flies, progeny from a cross between wild-type flies and flies containing the DNA depicted as a circle in panel A above, to all be phenotypically normal. However, they actually found female albino flies in these crosses. When these white flies were crossed to wild-type males they found that almost 100% of those offspring were white, although one would expect only the males to be. So their MCR approach clearly worked, and in a way it violates the age-old rules of Mendelian inheritance.
In their conclusion they stated that MCR may, “provid[e] a potent gene drive system for delivery of transgenes in disease vector or pest populations, and potentially serv[e] as a disease-specific delivery system for gene therapy strategies”. Gene drive “involves stimulating biased inheritance of particular genes to alter entire populations of organisms” (Pennisi (2014)). This approach may rapidly disseminate mutations that inactivate virulent traits in various disease vectors, such as the Anopheles mosquito, which acts as a vector for the malaria parasite Plasmodium falciparum.
Gantz and Bier also wrote that they are, “keenly aware of the substantial risks associated with this highly invasive method. Failure to take stringent precautions could lead to the unintentional release of MCR organisms into the environment”. In a comment on the paper their security precautions are mentioned: the flies are kept in three layers of tubes and boxes, locked behind five doors operated by fingerprint recognition to ensure they do not escape into the wild. However, just as Baltimore et al. (2015) called for caution and open discussions concerning CRISPR applications with regards to (human) genome-editing in general, Esvelt et al. (2014) demanded extreme caution in the use of CRISPR-based gene drive systems. They especially emphasised the need to both molecularly and ecologically contain these systems: at the molecular level, for example, the targeted gene must not be present in wild species; at the ecological level experiments should only be attempted in inhospitable environments where organisms will not easily find mating partners. Their hesitation to fully embrace this new approach is understandable. For example, what if the guide RNA becomes mutated and the nuclease starts targeting other genes as well? Furthermore, such gene drives employed by the government of a country, for instance, to combat a disease vector will almost inevitably cross political borders. If introduced into endangered species by accident they could eradicate them entirely. It seems to me that it will be quite difficult to test such approaches (in the manner of a clinical trial, say) without actually conducting the experiment in the wild. And somehow inexplicably these articles entirely slipped under my radar, whereas the recent human genome-editing experiment caused much more uproar although it is arguably a lot less prone to “spiral out of control”.
Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348: 36-38
Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014) Concerning RNA-guided gene drives for the alteration of wild populations DOI:10.7554/eLife.03401
Gantz VM, Bier E (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science 348: 442-444
Pennisi, E (2014) U.S. researchers call for greater oversight of powerful genetic technology. Science Insider: http://news.sciencemag.org/biology/2014/07/u-s-researchers-call-greater-oversight-powerful-genetic-technology. Accessed May 3rd 2015.