Advances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

The final body size of any given individual underlies both genetic and environmental constraints. Both mammals and insects use target of rapamycin (TOR) and insulin signaling pathways to coordinate growth with nutrition. In holometabolous insects, the growth period is terminated through a cascade of peptide and steroid hormones that end larval feeding behavior and trigger metamorphosis, a nonfeeding stage during which the larval body plan is remodeled to produce an adult. This irreversible decision, termed the critical weight (CW) checkpoint, ensures that larvae have acquired sufficient nutrients to complete and survive development to adulthood. How insects assess body size via the CW checkpoint is still poorly understood on the molecular level. We show here that the Drosophila transcription factor Snail plays a key role in this process. Before and during the CW checkpoint, snail is highly expressed in the larval prothoracic gland (PG), an endocrine tissue undergoing endoreplication and primarily dedicated to the production of the steroid hormone ecdysone. We observed two Snail peaks in the PG, one before and one after the molt from the second to the third instar. Remarkably, these Snail peaks coincide with two peaks of PG cells entering S phase and a slowing of DNA synthesis between the peaks. Interestingly, the second Snail peak occurs at the exit of the CW checkpoint. Snail levels then decline continuously, and endoreplication becomes nonsynchronized in the PG after the CW checkpoint. This suggests that the synchronization of PG cells into S phase via Snail represents the mechanistic link used to terminate the CW checkpoint. Indeed, PG-specific loss of snail function prior to the CW checkpoint causes larval arrest due to a cessation of endoreplication in PG cells, whereas impairing snail after the CW checkpoint no longer affected endoreplication and further development. During the CW window, starvation or loss of TOR signaling disrupted the formation of Snail peaks and endocycle synchronization, whereas later starvation had no effect on snail expression. Taken together, our data demonstrate that insects use the TOR pathway to assess nutrient status during larval development to regulate Snail in ecdysone-producing cells as an effector protein to coordinate endoreplication and CW attainment.

Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post‐transcriptional gene regulation.