Drosophila knock ins in a month without DNA manipulation.

A normal functioning nervous system is crucial for organismal health and dysfunction in parts of the nervous system can affect movement, memory formation and mood. Identification of genes responsible for inherited neuronal dysfunction permits to study their function in model organisms. Drosophila melanogaster is renowned for its powerful genetic tools for studying neurological diseases and allows investigation of these genes’ function in a physiological in vivo context (Bier, 2005). Most human disease genes have at least one functional ortholog in flies and transgenic expression of human disease genes can recapitulate features of the disease (Feany and Bender, 2000). Moreover many discoveries in the fruit fly led to novel insights in vertebrate biology (Bellen et al., 2010). Therefore Drosophila is the model system to dissect the function of human disease genes. More and more groups that are studying neuronal degeneration are collaborating with Drosophila groups and incorporate Drosophila genetics as an integral part of their scientific effort to study the function of their gene of interest and elucidate its molecular mechanisms (Laridon et al., 2008; Morais et al., 2009; Simpson et al., 2009; Coen et al., 2012). However so far Drosophila models are either loss-of-function models or models based on over/miss-expression and therefore do not necessarily recapitulate the exact function of the genes/mutations that are causing the disease. Therefore the ideal situation would be to knock in disease genes (that harbor pathological mutations) in the endogenous fly (ortholog) locus, providing endogenous expression levels in the relevant tissues.

Scientific aim

To understand the function of genes involved in a given disease, the fruit fly Drosophila melanogaster is often used. Its popularity comes from the power of its genetic toolkit. Different reverse genetic approaches have been developed to generate mutants; however the "cleanest" way to induce mutants is homologous recombination (HR), where the wild type endogenous locus is replaced by a mutant allele (Rong and Golic, 2000). HR thus allows researchers to create null alleles or knock in modified (mutant) versions of the gene studied. HR is at present not widely used in the fruit fly field as it is labor intensive and has a low efficiency (Gong and Rong, 2003). My recent work (Choi et al., 2009) significantly improved the way fruit fly researchers can generate multiple knock in events in their gene of interest by developing IMAGO, a technology that allows for recombinase mediated cassette exchange (RMCE) in the endogenous locus of a Drosophila gene. While a single HR event is still needed in IMAGO, subsequent gene replacements in the same locus (e.g. to create an allelic series, or to knock in multiple ‘diseased versions’ of a gene) are very efficient. Building on this expertise, here I propose to develop novel ground-breaking technology that will enable to create an RMCE-ready knock in allele in less than a month and –importantly- without the need for cloning pieces of DNA into targeting vectors or the need to inject such constructs in fruit fly embryos. If successful, this technology will revolutionarize fruit fly genetics as it will enable to create in a very easy fashion an RMCE-ready allele of virtually any gene in the fly genome (including disease-related genes) simply by crossing flies.

Methodology

Zinc finger nucleases (ZFN) have been developed as powerful tools to increase HR efficiency (Bibikova et al., 2003). ZFN are made of the catalytic domain of FokI fused to ZF repeat modules, specific DNA binding modules, custom combined to target unique stretches of genomic sequence. These enzymes introduce gene specific double strand breaks (DSB) that can be used to manipulate the genome at specific locations (Bibikova et al., 2002). Embryo injection of specific ZFN mRNAs against the gene of interest and a plasmid with target DNA generated recombinants with efficiencies up to 95% (Beumer et al., 2008) (compared to <1% using the classical approach). While clearly very efficient, the technology suffers from the fact that new ZFNs need to be developed (and tested) for each gene one wishes to target; a time consuming and costly endeavor.

Here we propose to use ZFN mediated gene replacement to generate one unified efficient tool that allows knock in in almost every gene in the Drosophila genome. Our strategy is based on generating a ZFN that recognizes a P-element transposon sequence. Making a DSB in P-element allows targeting loci located nearby a given P-element. Stock centers maintain tens of thousands of Drosophila stocks harboring molecularly mapped P-elements, and nearly every gene is located close (<5kb) to a given P-element (Bellen et al., 2011). Combining P-element insertions as molecular tags with ZFN technology would allow targeting most genes in the fly genome. Our strategy incorporates a RMCE cassette; thus, once a gene has been targeted, additional alleles of this gene can be obtained. By creating only once a collection of transgenic flies that express the Zn fingers and bear the P-element targeting cassette, there will be no need to inject animals to target a locus of interest as the Zn fingers and targeting constructs can be expressed/activated using inducible promotors. Crosses between fruit flies that harbor the P element and flies expressing the Zn fingers and targeting construct will enable to replace the locus of interest with the RMCE cassette. In subsequent steps this cassette can be targeted to express mutant forms of the gene, with a single injection step needed. As a proof of concept, in this proposal we will target several PD related genes (Mandemakers et al., 2007) in the fruit fly (parkin, pink1, dLRRK, dj-1α, dj-1β and omi) and knock in the clinical mutant alleles in a second step. If successful, this methodology will create a valuable toolkit to study PD in a genetic model organism and the method can easily be expanded to all other genes of the Drosophila genome.

Proof-of-principle: ZFN against yellow, a common marker in P-elements are available and provide us a fast means to test the ease to target the yellow+ P-elements with IMAGO cassettes. Later a ZFN against white, another common marker of transposable elements, will be generated and used for high throughput replacements of all (PD related) genes.

Perspectives: Our novel approach using advanced molecular genetic techniques in Drosophila will, if successful, allow us generate novel, better, models of Parkinson’s disease in Drosophila but also to target virtually every gene in the genome with an RMCE cassette simply by crossing flies. The methodology that I plan to develop will enable researchers to perform large scale structure-function analyses in a very clean and controlled genetic

background and will allow the development of large collections of mutations in groups of genes in a ‘unified’ manner.