Using single-cell RNA sequencing to generate predictive cell-type-specific split-GAL4 reagents throughout development.

Abstract

Cell-type-specific tools facilitate the identification and functional characterization of the distinct cell types that form the complexity of neuronal circuits. A large collection of existing genetic tools in <i>Drosophila</i> relies on enhancer activity to label different subsets of cells and has been extremely useful in analyzing functional circuits in adults. However, these enhancer-based GAL4 lines often do not reflect the expression of nearby gene(s) as they only represent a small portion of the full gene regulatory elements. While genetic intersectional techniques such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resources to screen through combinations of enhancer expression patterns. Here, we use existing developmental single-cell RNA sequencing (scRNAseq) datasets to select gene pairs for split-GAL4 and provide a highly efficient and predictive pipeline (scMarco) to generate cell-type-specific split-GAL4 lines at any time during development, based on the native gene regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines or by CRISPR knock-in. We use the developing <i>Drosophila</i> visual system as a model to demonstrate the high predictive power of scRNAseq-guided gene-specific split-GAL4 lines in targeting known cell types, annotating clusters in scRNAseq datasets as well as in identifying novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to any other <i>Drosophila</i> tissue. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource for the <i>Drosophila</i> community.