Analysis of genetic editing and expansion efficiency on isogenic cellular models subjected to CRISPR/Cas9 methodologies

Abstract

Introduction: Rare de novo loss-of-function (LoF) genetic variants in genes encoding genic expression regulators compose risk factors commonly associated to neurodevelopmental diseases (NDDs). POGZ represents a significant model within this gene class, so that rare variants on this gene are associated to White-Sutton syndrome.Objectives: Establishment of isogenic models of human induced pluripotent stem cells (hiPSC) with one LoF variant in POGZ observed in patients and evaluation of the functional consequences of this variant, as well as the evaluation of the efficiency of the methodologies used for genetic edition.Methods: The CRISPR/Cas9 genomic engineering methodology will be employed for the introduction of the variants into hiPSC using two gRNAs for the generation of gene deletion and, thereby, a bona fide of LoF model, and Prime Editing to generate patient-specific LoF variants.Expected Results: Evaluation of functional consequences will enable the identification of similarities between the introduced cellular phenotypes variants, allowing assessment of whether the variant found in patients acts through an haploinsufficiency mechanism. The efficiency evaluation of the applied genetic methodologies should indicate a higher efficiency associated with the Prime Editing methodology, employed for the patients' specific variant, in comparison with CRISPR/Cas9, used for the gene deletion insertion.