Garcia MF, Retallick-Townsley K, Pruitt A, Davidson E, Balafkan N, Warrell J, Huang TC, Kibowen A, Chu Z, Dai Y, Fitzpatrick SE, Meng R, Sen A, Cohen S, Livoti O, Khan S, Becker C, E Silva ALT, Liu J, Dossou G, Cheung J, Liu S, Ghorbani S, Deans PJM, DeCiucis M, Emani P, Gao H, Shen H, Gerstein M, Wang Z, Huckins LM, Hoffman EJ, Brennand K.

Links:
bioRxiv PREPRINT (doi.org/10.1101/2024.08.23.609190)
Datasets used for external validation of random forest models on GEO (GSE200774) and on Synapse (syn27819129)

Abstract:

Over three hundred and seventy-three risk genes, broadly enriched for roles in neuronal communication and gene expression regulation, underlie risk for autism spectrum disorder (ASD) and developmental delay (DD). Functional genomic studies of subsets of these genes consistently indicate a convergent role in neurogenesis, but how these diverse risk genes converge on a smaller number of biological pathways in mature neurons is unclear. To uncover shared downstream impacts between neurodevelopmental disorder (NDD) risk genes, here we apply a pooled CRISPR approach to contrast the transcriptomic impacts of targeting 29 NDD loss-of-function genes across human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Points of convergence vary between the cell types of the brain and are greatest in mature glutamatergic neurons, where they broadly target not just synaptic and epigenetic, but unexpectedly, mitochondrial biology. The strongest convergent networks occur between NDD genes with common co-expression patterns in the post-mortem brain, biological annotations, and clinical associations, suggesting that convergence may one-day inform patient stratification and treatment. Towards this, ten out of eleven drugs tested that were predicted to reverse convergent signatures in human cells and/or arousal and sensory processing behaviors in zebrafish ameliorated at least one behavioral phenotype in vivo. Altogether, robust convergence in post-mitotic neurons represents a clinically actionable therapeutic window.

Authors:

Kang B, Murphy M, Ng CW, Leventhal MJ, Huynh N, Im E, Danquah S, Housman DE, Nehme R, Farhi SL, Fraenkel E.

Links:

bioRxiv PREPRINT (doi.org/10.1101/2025.02.19.639023)
GitHub CellPHIE

Abstract:

Genomic screens and GWAS are powerful tools for identifying disease-modifying genes, but it is often challenging to understand the pathways by which these genes function. Here, we take an integrated approach that combines network analysis and an imaging-based pooled genetic perturbation study to examine modifiers of Huntington’s disease (HD). The computational analysis highlighted several genes in a subnetwork enriched for modifiers of neuronal development and morphology. To test the functional roles of these genes, we developed an experimental pipeline that allows pooled CRISPRi KD of 21 genes in human iPSC-derived neurons followed by optical analysis of genotypes, neuronal arborization, multiplexed pathway activity and morphological fingerprint readout. This approach recovered known genes involved in morphology and confirmed unexpected links from the network between several genetic modifiers of HD and morphology. Our approach overcomes challenges in pooled measurement of neuronal function and health and could be adapted for other phenotypes in HD and other neurological diseases.

Authors:

Hanwen Zhang, Ada McCarroll, Lilia Peyton, Sol Díaz de León-Guerrerro, Siwei Zhang, Prarthana Gowda, David Sirkin, Mahmoud ElAchwah, Alexandra Duhe, Whitney G. Wood, Brandon Jamison, Gregory Tracy, Rebecca Pollak, Ronald P. Hart, Carlos N. Pato, Jennifer G. Mulle, Alan R. Sanders, Zhiping P. Pang, Jubao Duan

Links:

Stem Cell Reports Paper  (doi.org/10.1016/j.stemcr.2024.08.003)
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.

Authors:

Loϊc Binan, Aiping Jiang, Serwah A. Danquah, Vera Valakh, Brooke Simonton, Jon Bezney, Robert T. Manguso, Kathleen B. Yates, Ralda Nehme, Brian Cleary, Samouil L. Farhi

Links:

Paper – (doi.org/10.1016/j.cell.2025.02.012)
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 they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.