SSPsyGene Data
2023
Simultaneous CRISPR screening and spatial transcriptomics reveals intracellular, intercellular, and functional transcriptional circuits
Loϊc Binan, Serwah Danquah, Vera Valakh, Brooke Simonton, Jon Bezney, Ralda
Nehme, Brian Cleary, Samouil L Farhi.
Links:
Paper – (doi.org/10.1101/2023.11.30.569494)
Perturb-FISH protocol – (dx.doi.org/10.17504/protocols.io.6qpvr356bvmk/v1)
GitHub FR-Perturb
GitHub Perturb-FISH
GEO Accession – (GSE221321)
Brain Image Library Landing Page – (https://doi.org/10.35077/ace-gem-get)
BIL Download Data Directory
BIL Metadata API
Abstract:
Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.
2024
Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs
Links:
Stem Cell Reports Paper
RNA-seq Data – (GSE262442)
Reported data and code used during analysis – ( zenodo.13273149)
Abstract:
Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.