Author ORCID Identifier
Document Type
Dissertation
Date of Award
2026
Degree Name
Doctor of Philosophy (PhD)
Department
Basic Biomedical Science
First Advisor
Louis-Jan Pilaz
Abstract
Many neurodevelopmental disorders occur due to perturbations in prenatal brain development. More genetic variants associated with neurodevelopmental disorders are continuously being identified. However, there is a lack of understanding of the pathogenic mechanisms. Here, we describe a novel application of the technique Breasi-CRISPR in modeling specific patient variants in the embryonic mouse brain. Breasi-CRISPR is a highly efficient system combining in utero electroporation with CRISPR-CAS9 genome editing. We hypothesized that Breasi-CRISPR would be sufficient at inserting patient analogous variants into endogenous loci to be used as a new paradigm for modeling neurodevelopmental disorders. We aimed to test if Breasi-CRISPR could recapitulate patient phenotypes and enable investigation of the molecular mechanisms underlying neurodevelopmental disorders. First, we modeled patient variants in MAP1B which was recently associated with periventricular nodular heterotopia, associated with dysregulated neuron migration. We introduced two patient analogous variants into the embryonic cortex via Breasi-CRISPR. Both variants caused migration delays of different severity. We were able to demonstrate that these variants produced a truncated protein; however, this protein fragment did not act as a dominant negative. Next, we modeled a missense patient variant in CCND2 associated with megalencephaly postaxial polydactyly polymicrogyria hydrocephalus syndrome, characterized by excessive proliferation. Following insertion of the most common patient variant, we saw an increase in cells in S phase and expansion of the number of neural progenitor cells, mirroring excessive proliferation phenotype seen in patients. Consistent with previous reports, we were also able to demonstrate higher levels of cyclin D2 in the electroporated region. RNAseq analysis demonstrated that the patient variant caused downregulation of genes associated with we have used Breasi-CRISPR to model two types of patient variants in two neurodevelopmental disorders with widely different patient phenotypes. In both cases, Breasi-CRISPR was sufficient to recapitulate the phenotype of the patients and enabled downstream analyses of the pathogenic mechanism. We predict that Breasi-CRISPR will become an enabling tool to accelerate the investigation of how patient variants lead to neurodevelopmental disorders.
Subject Categories
Genetics | Neuroscience and Neurobiology
Keywords
CRISPR-CAS9 genome megalencephaly postaxial polydactyly polymicrogyria hydrocephalus syndrome
Number of Pages
97
Publisher
University of South Dakota
Recommended Citation
Kittock, Claire Mae, "Accelerating the discovery of the genetic and molecular bases of neurodevelopmental disorders in vivo using Breasi-CRISPR" (2026). Dissertations and Theses. 397.
https://red.library.usd.edu/diss-thesis/397