CRISPR/Cas9 gene editing in Drosophila via visual selection in a summer classroom.

CRISPR/Cas9 methods are a powerful in vivo approach to edit the genome of Drosophila melanogaster. To convert existing Drosophila GAL4 lines to LexA driver lines in a secondary school classroom setting, we applied the CRISPR-based genetic approach to a collection of Gal4 'driver' lines. The integration of the yellow+ coat color marker into homology-assisted CRISPR knock-in (HACK) enabled visual selection of Gal4-to-LexA conversions using brightfield stereo-microscopy available in a broader set of standard classrooms. Here, we report the successful conversion of eleven Gal4 lines with expression in neuropeptide-expressing cells into corresponding, novel LexA drivers. The conversion was confirmed by LexA- and Gal4-specific GFP reporter gene expression. This curriculum was successfully implemented in a summer course running 16 hours/week for seven weeks. The modularity, flexibility, and compactness of this course should enable development of similar classes in secondary schools and undergraduate curricula, to provide opportunities for experience-based science instruction, and university-secondary school collaborations that simultaneously fulfill research needs in the community of science.

Simplified homology-assisted CRISPR for gene editing in Drosophila.

In vivo genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 generates powerful tools to study gene regulation and function. We revised the homology-assisted CRISPR knock-in method to convert Drosophila GAL4 lines to LexA lines using a new universal knock-in donor strain. A balancer chromosome-linked donor strain with both body color (yellow) and eye red fluorescent protein (RFP) expression markers simplified the identification of LexA knock-in using light or fluorescence microscopy. A second balancer chromosome-linked donor strain readily converted the second chromosome-linked GAL4 lines regardless of target location in the cis-chromosome but showed limited success for the third chromosome-linked GAL4 lines. We observed a consistent and robust expression of the yellow transgene in progeny harboring a LexA knock-in at diverse genomic locations. Unexpectedly, the expression of the 3xP3-RFP transgene in the "dual transgene" cassette was significantly increased compared with that of the original single 3xP3-RFP transgene cassette in all tested genomic locations. Using this improved screening approach, we generated 16 novel LexA lines; tissue expression by the derived LexA and originating GAL4 lines was similar or indistinguishable. In collaboration with 2 secondary school classes, we also established a systematic workflow to generate a collection of LexA lines from frequently used GAL4 lines.

A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase.

We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten constant (Km ) of 15.89 µm. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analogue. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analogue.

Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAFV600E mutation bearing metastatic melanoma cells.

Treatment with vemurafenib, a potent and selective inhibitor of mitogen-activated protein kinase signaling downstream of the BRAFV600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant metastatic melanomas. We have conducted high-throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib-resistant melanoma cell lines, A2058R and A375R (containing the BRAFV600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E-BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib-resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug-resistant melanomas harboring BRAF V600E mutations.