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Identification of convergent molecular pathways in autism spectrum disorder by in utero genome editing and transcriptional profiling of cortical projection neuron subtypes

English title Identification of convergent molecular pathways in autism spectrum disorder by in utero genome editing and transcriptional profiling of cortical projection neuron subtypes
Applicant Platt Randall Jeffrey
Number 175830
Funding scheme Project funding (Div. I-III)
Research institution Computational Systems Biology Department of Biosystems, D-BSSE ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Neurophysiology and Brain Research
Start/End 01.09.2018 - 31.08.2022
Approved amount 700'000.00
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All Disciplines (5)

Discipline
Neurophysiology and Brain Research
Cellular Biology, Cytology
Embryology, Developmental Biology
Genetics
Molecular Biology

Keywords (6)

RNA sequencing; Neocortical development; Cortical projection neurons; Genome editing; Mouse models; Autism

Lay Summary (German)

Lead
Entschlüsselung der genetischen und zellulären Grundlagen von Autismus
Lay summary

Die Autismus-Spektrum-Störung (ASS) stellt eine vielfältige Sammlung verwandter Erkrankungen dar, die einen von einhundert Menschen weltweit betrifft. Aktuelle Studien über die genetischen Ursachen, bringen mehr als 1000 Gene in Verbindung mit der Erkrankung. Darüber hinaus gibt es Anzeichen dafür, dass eine Klasse von Neuronen im Gehirn, die als Projektionsneuronen bezeichnet werden, ebenfalls mit der Störung in Zusammenhang stehen. Obwohl das Wissen über möglicherweise beteiligte Gene und Zellen einen ersten Ansatz für die Untersuchung der Störung bietet, sind die zugrundeliegenden Ursachen von ASS und wie die Veränderungen in Genen und Zellen zu Verhaltenssymptomen bei menschlichen Patienten führen, weitgehend unbekannt. Das Ziel unseres Projekts besteht darin, eine Methode zu entwickeln, die es ermöglicht den Einfluss von Mutationen in jedem einzelnen dieser 1000 Gene auf die Genexpression in verschiedenen Projektionsneuronen zu untersuchen. Der Vergleich der resultierenden Genexpressionsprofile, kann es uns ermöglichen allgemeine zelluläre Störungen, welche die Grundlage für ASS bilden zu identifizieren, sowie spezifische zelluläre Störungen, die verschiedene Gruppen von ASS-Patienten widerspiegeln. Die Kenntnis über allgemeine und spezifische zelluläre Veränderungen kann einen Ausgangspunkt für ein besseres Verständnis der Störung bieten und zeigen, wie einzelne Veränderungen im Gehirn zu einer bestimmten Art von ASS führen. Letztendlich würde ein besseres Verständnis der genetischen und zellulären Grundlagen von ASS zu einer verbesserten Diagnose und einer präziseren Therapie für Patienten führen.

Direct link to Lay Summary Last update: 15.08.2018

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Employees

Abstract

Autism spectrum disorder (ASD) is a collection of phenotypically heterogeneous neurodevelopmental disorders affecting 1/100 individuals worldwide, and recent patient sequencing based studies have revealed over 1000 risk alleles. Evidence is mounting that suggests the neurobiological basis of ASD is associated with projection neurons (PNs) of the mammalian neocortex during midfetal development. Although these advancements provide an entry point into studying the disorder, the molecular underpinnings of ASD as well as how the disparate risk alleles converge onto a common set of pathways leading to similar behavioral phenotypes are largely unknown. To identify the convergent pathways underlying ASD, we will genetically perturb ASD risk genes in utero and directly compare the resulting molecular, cellular, and morphological phenotypes in cortical PN subtypes. Building on our previous work on mouse models of ASD, transcriptional profiling, and in vivo and in utero genome editing, we will: Aim 1 - Develop CRISPR-Cas9 reagents to perturb high confidence ASD risk genes in utero. We will develop and validate CRISPR-Cas9 reagents targeting mouse orthologs of high confidence ASD (hcASD) risk genes CHD8, DYRK1A, ARID1B, and POGZ by in utero electroporation (IUE) and genome editing analysis in mice. Methods enabling the rapid modeling of loss-of-function (LOF) variants in ASD risk genes in vivo will facilitate the systematic interrogation of the underlying mechanism(s) of ASD.Aim 2 - Investigate gene-edited cortical progenitors and projection neurons in situ to reveal shared phenotypes. In mice harboring LOF variants in select hcASD risk genes, we will investigate cortical progenitor and PN phenotypes, such as progenitor cell proliferation and differentiation as well as PN composition and lamination, respectively. This approach will pinpoint relevant cell types and phenotypes shared upon perturbation of select hcASD risk genes.Aim 3 - Characterize gene-edited cortical projection neuron subtypes by transcriptional profiling to identify shared ASD pathways. From gene-edited mice, we will purify cortical PN subtypes harboring LOF variants in select hcASD risk genes and perform transcriptional profiling of each population at high resolution. An integrated analysis across genotypes will allow us to distinguish gene-specific effects from shared pathways, enabling the identification of critical cell types and the convergent pathways underlying perturbation of select hcASD risk genes.Aim 4 - Characterize gene-edited neurons by pooled in utero perturbation and single cell transcriptional profiling to identify convergent ASD pathways. Using pooled lentiviral CRISPR libraries, we will perturb 100 probable and high confidence ASD risk genes in utero, such that single cells receive single perturbation, and subsequently perform single cell transcriptional profiling. After linking ASD risk gene perturbations with single cell transcriptional profiles, we will perform an integrated analysis to characterize the cell states and convergent molecular pathways shared upon perturbation of 100 ASD risk genes.The proposed studies will establish an in utero methodology for rapidly perturbing and phenotypically investigating meaningful cell types harboring LOF variants in ASD risk genes in vivo, thereby overcoming current limitations in ASD research involved with animal model generation and cross-model comparisons. By investigating multiple ASD risk genes and cell types in parallel, under identical experimental conditions, we will elucidate critical features underlying the neurobiological mechanism(s) of ASD. The methodologies developed here will be a valuable resource to the field and can be extended to any gene or gene set of interest.
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