GSE185022
Optogenetic perturbations of RNA expression in tissue space
Quantifying gene expression in space, for example by spatial transcriptomics, is essential for describing the biology of cells and their interactions in complex tissues. Perturbation experiments, at single-cell resolution and conditional on both space and time, are necessary for dissecting the molecular mechanisms of these interactions. To this aim, we combined optogenetics and CRISPR technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatial patterns. As a proof of principle, we optogenetically induced Sonic Hedgehog (SHH) signaling at a distinct spatial location within human neural organoids. This robustly induced known SHH spatial domains of gene expression – cell-autonomously and across the entire organoid. In principle, our approach can be used to induce or knock down RNAs from any gene of interest in specific spatial locations or patterns of complex biological systems. Overall design: Total RNA-seq for two replicates of HEK293T cells transfected with plasmids encoding: Rfx-Cas13d (CasRx), a TetON TagRFP reporter, and either a non-targeting guide RNA or an RFP-targeting RNA. 10X Visium sequencing data from 4 samples of human induced pluripotent stem cells (hiPSCs) stably expressing a light-inducible Cre/Lox system and a LoxP-dsRed-LoxP-SHH-NeonGreen cassette for light-inducible overexpression of SHH. The 4 samples are: non-induced cells and cells induced for 36, 48, 120h. Drop-seq single-cell RNA-seq data from 4 samples of 15-days old neural organoids, stably expressing a light-inducible Cre/Lox system and a LoxP-dsRed-LoxP-SHH-NeonGreen cassette for light-inducible overexpression of SHH. The samples are two replicates of controls (non-induced) and two replicates of SHH-induced organoids pools (3 organoids per pool).