Engineering the Drosophila Genome for Developmental Biology

Abstract

The recent development of transposon and CRISPR-Cas9-based tools for manipulating the fly genome in vivo promises tremendous progress in our ability to study developmental processes. Tools for introducing tags into genes at their endogenous genomic loci facilitate imaging or biochemistry approaches at the cellular or subcellular levels. Similarly, the ability to make specific alterations to the genome sequence allows much more precise genetic control to address questions of gene function.

Keywords: CRISPR-Cas9; Drosophila; genome engineering; protein tagging.

Figure 1

Genome engineering. (a–c) The Golic method for generating precise chromosomal deletions. (a) Two P elements, designated RS3 and RS5, are inserted at different locations (designated by a–d) on two homologous chromosomes and kept in separate fly lines. The elements contain a functional mini-white gene composed of multiple exons that for simplicity are drawn as grey or white boxes, representing 5′ and 3′ portions of the gene. There are two Frt sites (grey arrows) in each element, one of which is located within a mini-white exon. The elements differ in the position of the second Frt site and the orientation of the construct with respect to the P element ends (blue triangles). (b) Internal Flp-driven recombination between the Frt sites produces remnant forms of the white genes such that RS5r contains the 5′ end and RS3r the 3′ end with the intronic FRT site remaining. Each of these remnant elements are generated in separate fly lines that are phenotypically white eyed. (c) RS5r and RS3r elements are brought together in trans in a fly along with a source of Flp recombinase. FLP-mediated recombination between the elements produces a reconstituted functional white gene and the intervening genomic DNA is deleted. The reciprocal event creates a tandem duplication of the deleted segment, separated by an FRT site, but no white gene. (d) The PhiC31 system. A transposon (blue triangles mark the transposon ends) carrying a marker gene for genetic tracking and an attP site is inserted into the genome. Providing an attB containing plasmid with a gene or sequence of interest, in this case GFP, and a source of PhC31 integrase results in high efficiency integration of the plasmid into the genomic location. (e) Recombinase-mediated cassette exchange (RMCE). A transposon (blue triangles mark the transposon ends) carrying a marker gene flanked by attP site is inserted into the genome. Providing a plasmid with a gene or sequence of interest, in this case GFP, flanked by attB sites and a source of PhC31 recombinase results in high efficiency replacement of the genomic marker with the sequence of interest. With RMCE the inverted orientation of the attP and attB sites is critical for producing the desired exchange.

Figure 2

Protein Trapping. (a–c) piggyBac transposon-based protein trapping. (a) The pigP protein trap element used in [47]: the transposon ends (blue triangles) flank a genetic marker gene (white) and an artificial exon, which in this case contains the coding sequence for enhanced green fluorescent protein (EGFP) along with StrepII and 3XFLAG tags between splice donor and splice acceptor sites (blue circles). (b) Insertion of the transposon into an intron of a protein coding gene (represented by lines separating the yellow boxes) allows the possibility of splicing the artificial exon into the gene transcript. (c) If the transcript carrying the artificial exon is translated a tagged protein is generated. (d,e) The Minos-mediated integration cassette (MiMIC) System. (d) A Minos-based transposon with transposable element (TE) ends indicated by blue triangles, contains two attP sites in inverted orientation (blue diamonds) flanking a gene trap cassette with a splice acceptor (blue circle), stop codons in all three reading frames (red box), a fluorescent marker (EGFP) followed by a polyadenylation signal (blue box) and a genetic marker (yellow). The sequences internal to the attP sites may be replaced via a RMCE reaction by providing a donor sequence flanked by attB sites and a source of phiC31 integrase. (e) A variety of different fluorescent reporters have been developed that can be used to introduce tags into genes with MiMIC insertions in coding introns via RMCE.

Figure 3

CRISPR Genome Engineering. (a) The wild-type Cas9 complex (light blue cloud) contains the Cas9 endonuclease and a guide RNA (gRNA) (blue) complementary to the target site adjacent to a PAM sequence (orange). The complex opens the DNA duplex and introduces a double strand break (red triangles). Repair by the non-homologous end joining (NHEJ) pathway may result in indel mutations whereas homology directed repair (HDR) in the presences of a donor template (green) generates insertions. (b) Using mutant Cas9n enzymes that make single strand cuts with two gRNAs (blue) direct the Cas9n complexes to make cuts (red triangles) separated by some distance. The gap may be repaired in the presence of a donor (green) to generate an insertion. (c) Dead Cas9 (dCas9) enzymes (which are unable to cleave DNA) are fused with FokI nuclease monomers (orange). When two gRNAs (blue) some distance apart are used, dCas9-FolkI monomers are brought into proximity allowing the FokI to dimerise and cleave in between. The resulting gap may be repaired in the presence of a donor (green) to generate an insertion. (d) Diagram of donor cassette for direct addition of a protein tag to the C-terminal of a coding exon. The donor DNA contains part of the exon sequence with a biochemistry tag and yellow fluorescent protein (YFP) in frame, followed by a right hand end homology arm (RHA) that can mediate the type of insertion event shown in (a). (e) Diagram of a donor sequence used to introduce biochemistry tags to the C-terminal of a coding exon along with a removable marker. The donor DNA contains part of the exon sequence with a biochemistry tag in frame. This is followed by an eye expressed red fluorescent protein (RFP) cassette flanked by LoxP sites, which are downstream of the splice donor site and thus within an intron, then a right hand homology arm. Once a verified insertion has been recovered, tracked by the acquisition of RFP, the RFP is removed by exposing to Cre recombinase and recovering flies who have lost RFP expression. The resulting lines have the tagged exon and a single intronic LoxP site.