Abstract
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a misactivation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.
Highlights
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control
We designed a panel of 13 single guide RNAs (sgRNAs) (18–20 bp) targeted to candidate upstream promoter and enhancer regions of 7 developmental genes in the Drosophila genome, namely dpp, engrailed, eve, hairy, hid, rad[51] and reaper (Fig. 2A)
In the pre-CRISPR era, it required the exploitation of complex fly genetics and knowledge of unique biological aspects of the Drosophila glass gene
Summary
The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. CRISPR gene activation (CRISPRa) uses a nuclease-deficient, deactivated Cas[9] (dCas9) protein, fused to a transactivation domain which recruits the basal transcriptional machinery to the site of sgRNA complementarity[12,13,14,15,16,17] This expanded CRISPR toolset allows for the exploration of more radical genetic engineering concepts one of which is the design of artificial reproductive isolation and the generation of synthetic species. In this way synthetic lethality is triggered by the transgene in hybrids that result from a cross between modified individuals and those of the naive genetic background This approach is in principle generalizable and could work in any tractable sexually reproducing organism, because it circumvents the need to research and employ species-specific modes of incompatibility or interfere with endogenous regulatory pathways in order to engineer isolation. This work highlights that the majority of experimental work utilizing dCas[9] with a view to application remains cell-based[22,23] with its potential in complex systems largely unexplored
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