Biology subjects

Page, N. F.

Publications and source records attributed to Page, N. F..

3 recordsLinked to original sources

bioRxiv2023

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

genomics↗

bioRxiv2022

Autism-associated transcriptional regulators target shared loci proximal to brain-expressed genes.

Many autism spectrum disorder (ASD)-associated genes act as transcriptional regulators (TRs). ChIP-seq was used to identify the regulatory targets of ARID1B, BCL11A, FOXP1, TBR1, and TCF7L2, ASD-associated TRs in the developing human and mouse cortex. These TRs shared substantial overlap in the binding sites, especially within open chromatin. The overlap within a promoter region, 1-2,000bp upstream of transcription start site, was highly predictive of brain expressed genes. This signature was observed at 96 out of 102 ASD-associated genes. In vitro CRISPRi against ARID1B and TBR1 delineated downstream convergent biology in mouse cortical cultures. After eight days, NeuN+ and CALB+ cells were decreased, GFAP+ cells were increased, and transcriptomic signatures correlated with the postmortem brain samples from individuals with ASD. We suggest functional convergence across five ASD-associated TRs leads to shared neurodevelopmental outcomes of haploinsufficient disruption.

genomics↗

bioRxiv2020

Alterations in retrotransposition, synaptic connectivity, and myelination implicated by transcriptomic changes following maternal immune activation in non-human primates

BackgroundMaternal immune activation (MIA) is a proposed risk factor for multiple neurodevelopmental and psychiatric disorders, including schizophrenia. However, the molecular and neurobiological mechanisms through which MIA imparts risk for these disorders remain poorly understood. A recently developed nonhuman primate model of exposure to the viral mimic poly:ICLC during pregnancy shows abnormal social and repetitive behaviors and elevated striatal dopamine, a molecular hallmark of human psychosis, providing an unprecedented opportunity for mechanistic dissection. MethodsWe performed RNA-sequencing across four psychiatrically-relevant brain regions (prefrontal cortex, anterior cingulate, hippocampus, and primary visual cortex) from 3.5-4-year old male MIA-exposed and control offspring--an age comparable to mid adolescence in humans. ResultsWe identify 266 unique genes differentially expressed (DE) in at least one brain region with the greatest number observed in hippocampus. Co-expression networks identified region-specific alterations in synaptic signaling and oligodendrocytes. Across regions, we observed temporal and regional differences, but transcriptomic changes were largely similar across 1st or 2nd trimester MIA exposures, including for the top DE genes--PIWIL2 and MGARP. In addition to PIWIL2, several other known regulators of retrotransposition, as well as endogenous transposable elements were dysregulated in MIA offspring. ConclusionsTogether, these results begin to elucidate the brain-level molecular mechanisms through which MIA may impart risk for psychiatric disease.

neuroscience↗