A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues

Abstract

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.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET

“…However, homozygous IRP1A FCF flies were not viable on regular fly media, indicating that the inserted FRT sites had generated a loss-of- IRP1A -function allele. Therefore, we employed a second CRISPR strategy, where we crossed flies that specifically expressed CAS9 in the PG 20 to flies expressing two gRNAs that targeted IRP1A (Supplementary Table 1). The resulting F1 progeny also displayed PG-specific autofluorescence and 100% lethality on regular fly media (Fig.…”

A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion

“…To generate lines for tissue-specific CRISPR-based deletion of Hph and sima/HIF-1a , double guide RNAs were cloned into plasmid pCFD6 82,83 (Addgene #73915). The Hph construct should lead to the deletion of part of the genomic region encoding the dioxygenase domain common to all Hph isoforms, and the sima gRNAs target the region encoding that protein’s DNA-binding domain.…”

A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

“…Currently, CRISPR/Cas9 approaches allow scientists to precisely alter gene function via (i) classic CRISPR to introduce short INDELs, (ii) HR-based CRISPR for homology-based gene replacements or deletions, (iii) somatic CRISPR for conditional gene disruption, (iv) CRISPRi, (i = interference) to interfere with gene transcription, and (v) CRISPRa (a = activation) to upregulate gene activity. Studies have shown that it is possible to conditionally target genes of interest by exerting spatial and temporal control over Cas9 expression or using ligand-activated Cas9 variants [ 8 , 10 , 12 , 13 ]. The rapid advances in CRISPR technologies have made it a popular choice over earlier nuclease-based gene-editing approaches like meganucleases (MNs) [ 14 , 15 ], zinc finger nucleases (ZFNs) [ 16 – 18 ], and transcription activator-like effector nucleases (TALENs) [ 19 , 20 ].…”