An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

A gene-specific T2A-GAL4 library for Drosophila.

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes.

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.

The Drosophila gene disruption project: progress using transposons with distinctive site specificities.

The Drosophila Gene Disruption Project (GDP) has created a public collection of mutant strains containing single transposon insertions associated with different genes. These strains often disrupt gene function directly, allow production of new alleles, and have many other applications for analyzing gene function. Here we describe the addition of ∼7600 new strains, which were selected from >140,000 additional P or piggyBac element integrations and 12,500 newly generated insertions of the Minos transposon. These additions nearly double the size of the collection and increase the number of tagged genes to at least 9440, approximately two-thirds of all annotated protein-coding genes. We also compare the site specificity of the three major transposons used in the project. All three elements insert only rarely within many Polycomb-regulated regions, a property that may contribute to the origin of "transposon-free regions" (TFRs) in metazoan genomes. Within other genomic regions, Minos transposes essentially at random, whereas P or piggyBac elements display distinctive hotspots and coldspots. P elements, as previously shown, have a strong preference for promoters. In contrast, piggyBac site selectivity suggests that it has evolved to reduce deleterious and increase adaptive changes in host gene expression. The propensity of Minos to integrate broadly makes possible a hybrid finishing strategy for the project that will bring >95% of Drosophila genes under experimental control within their native genomic contexts.

Developmental and tissue-specific accumulation pattern for the Drosophila melanogaster TART ORF1 protein.

The TART, HeT-A, and TAHRE families of Drosophila non-LTR retrotransposons specifically retrotranspose to telomeres to maintain telomeric DNA. Recent evidence indicates that an RNA interference mechanism is likely to regulate TART, HeT-A, and TAHRE retrotransposition, but the developmental and tissue-specific expression of telomeric retrotransposon proteins has not previously been investigated. We have generated antisera against TART ORF1 protein (ORF1p) and used these antisera to examine the pattern of TART ORF1p expression in Drosophila melanogaster. We detected TART ORF1p throughout most of development and observed particularly high levels of protein in late larval and pupal stages. In late-stage larvae, ORF1p accumulates in brain and imaginal discs tissues, rather than in terminally differentiated larval tissues. Accumulation of ORF1p in imaginal discs is intriguing, since TART antisense RNA has previously been detected in imaginal discs, and we discuss the implications of these findings for TART regulation.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons.

The Drosophila non-long terminal repeat (non-LTR) retrotransposons TART and HeT-A specifically retrotranspose to chromosome ends to maintain Drosophila telomeric DNA. Relatively little is known, though, about the regulation of their expression and their retrotransposition to telomeres. We have used rapid amplification of cDNA ends (RACE) to identify multiple transcription initiation and polyadenylation sites for sense and antisense transcripts of three subfamilies of TART elements in Drosophila melanogaster. These results are consistent with the production of an array of TART transcripts. In contrast to other Drosophila non-LTR elements, a major initiation site for sense transcripts was mapped near the 3' end of the TART 5'-untranslated region (5'-UTR), rather than at the start of the 5'-UTR. A sequence overlapping this sense start site contains a good match to an initiator consensus for the transcription start sites of Drosophila LTR retrotransposons. Interestingly, analysis of 5' RACE products for antisense transcripts and the GenBank EST database revealed that TART antisense transcripts contain multiple introns. Our results highlight differences between transcription of TART and of other Drosophila non-LTR elements and they provide a foundation for testing the relationship between exceptional aspects of TART transcription and TART's specialized role at telomeres.

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.