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High throughput genomic edition to investigate neurodevelopmental disorders associated with CREBBP using isogenic cellular models

Grant number: 23/01662-9
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2023
Effective date (End): December 31, 2023
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal Investigator:Mariana Moysés Oliveira
Grantee:Mayara Paschalidis
Host Institution: Associação Fundo de Incentivo à Pesquisa (AFIP). São Paulo , SP, Brazil
Associated research grant:21/09089-0 - High throughput genomic edition to investigate neurodevelopmental disorders using isogenic cellular models, AP.JP

Abstract

Introduction: Rare de novo loss of function (LoF) and missense variants in genes that encode gene expression regulators constitute risk factors commonly associated with neurodevelopmental diseases (NDD). CREBBP represents a great model within this class of genes, since several mutational mechanisms act in its association with NDD. While CREBBP haploinsufficiency, either by microdeletions in 16p13.3 or point mutations, are associated with Rubinstein-Taybi syndrome, microduplications in 16p13.3 and missense variants in a specific domain of the protein (i.e. ZNF) are related to Menke-Hennekam syndrome. Thus, it is hypothesized that these last variants act through a gain-of-function (GoF) mechanism. Aims: Establishment of isogenic models of human induced pluripotent stem cells (hiPSC) with LoF and ZNF missense variants in CREBBP and evaluation of the functional consequences of the introduction of these variants, as well as evaluation of the efficiency of the methodologies used for gene editing. Methods: The CRISPR-Cas9 genomic engineering methodology will be used for the introduction of the variants using two gRNAs to generate gene deletion and, thus, a bona fide model of LoF, and Prime Editing to generate patient-specific missense variants. These models will be differentiated in glutamatergic cortical neurons and evaluated by RNA-seq. Expected results: The evaluation of the functional consequences will allow the identification of discrepancies between the cellular phenotypes resulting from the introduced variants. Thus, it is expected that the LoF variant produces a cellular phenotype associated with CREBBP gene haploinsufficiency, while the missense variant produces cellular phenotype associated with the gene's GoF. Functional impacts of LoF and GoF should generate a mirror phenotype, that is, with fold change in opposite directions when compared to the wild-type genotype. The evaluation of the efficiency of the applied genetic editing methodologies will enable the confirmation of greater efficiency linked to the Prime Editing methodology, used for the missense variant, in comparison with the dual guide CRISPR/Cas9, related to the editing of the LoF variant.

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