Pooled CRISPRa/i Screening for Neurodevelopmental and Autism Gene Analysis

• Grant number: 24/09806-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: November 25, 2024
• End date: November 24, 2025
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Andréa Laurato Sertié
• Grantee: André Luiz Teles e Silva
• Supervisor: Kristen Brennand
• Host Institution: Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Sociedade Beneficente Israelita Brasileira Albert Einstein (SBIBAE). São Paulo , SP, Brazil
• Institution abroad: Yale School of Medicine (YSM), United States
• Associated to the scholarship: 22/15760-0 - Study of molecular and cellular mechanisms involved in the oligogenic inheritance of Autism Spectrum Disorder, BP.DR

Abstract

Autism Spectrum Disorder (ASD) has a complex genetic architecture, with some patients carrying rare, deleterious variants in multiple risk genes, suggesting oligogenic inheritance. However, little is known about how these variants interact and converge on causative neurobiological pathways. Recently, we have identified in one Brazilian proband with ASD and macrocephaly, referred to as F2688-1, rare compound heterozygous missense variants in the Reelin gene (RELN) and a de novo splice site variant in the calcium (Ca2+) channel Cav3.2 gene (CACNA1H). Using hiPSCs-derived neural progenitor cells from patient F2688-1 and controls, we found that the variant in Cav3.2 leads to increased Ca2+ influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling by enhancing the degradation of downstream targets of Reelin. Finally, analysis of the sequencing data from two ASD cohorts - a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD - revealed a significant increased burden of simultaneous risk variants in both alleles of a gene in the Reelin pathway and in one allele of a gene for a Ca2+ channel in ASD. However, there are many unanswered questions about the pathomechanism behind these co-occurring variants. This BEPE proposal aims to fill this knowledge gap by using recent advancements in CRISPR-Cas9 and hiPSC-derived disease modeling. Specifically, we will use CRISPR activation and interference (CRISPRa/i) techniques to modulate the expression of CACNA1H and RELN genes in hiPSC-derived neurons and, then, correlate the expression of these genes to specific neuronal phenotypes. This integrated approach promises to study the pathophysiology of ASD more comprehensively, creating models that mimic genetic mutations with isogenic controls. These protocols can be adapted for other genes, allowing assessment of the phenotypic impact of candidate genes associated with neurodevelopmental or brain disorders