Honors Theses
Date of Award
Spring 5-8-2026
Document Type
Undergraduate Thesis
Department
Biomedical Engineering
First Advisor
Joshua Bloomekatz
Second Advisor
Kristine Willett
Third Advisor
Bradley Jones
Relational Format
Dissertation/Thesis
Abstract
Congenital heart defects (CHDs) are the most common class of birth defects. Despite advances in genomic sequencing, functional characterization of candidate genes remains a major challenge. This study aimed to develop two scalable CRISPR-based screening platforms to interrogate gene function during zebrafish cardiac development. The first CRISPR/Cas9 platform incorporates guide design, multiplexed ribonucleoprotein injections, and sequencing-based validation of target loci. RNA guides targeting genes involved in O-GlcNAc signaling, along with control genes (fn1a and gfp) were designed using the algorithm CHOPCHOP; selection criteria included predicted efficiency and genomic location. To ensure compatibility between guide sequences and the experimental zebrafish strain, seventeen target loci were amplified from pooled wild-type embryos and sequenced using Illumina sequencing technology. A custom bioinformatics pipeline was implemented to assess quality, sequence variation, mapping efficiency, and coverage across all targets. This analysis revealed high data quality, with an average mapping rate of approximately 96.5% and strong enrichment of reads within target amplicons (~84.3% on target). Coverage across all loci exceeded the 500x threshold required for detection of low-frequency variants. Minimal sequence variation between samples and the reference genome was observed, with only a single locus exhibiting polymorphisms. However, this polymorphism did not overlap guide RNA binding regions. These findings confirm that the selected guides are compatible with the experimental strain and suitable for downstream genome editing applications. As this RNP-based platform is limited to twenty-one guides (seven genes with 3 targets) at a time, a Cas12a-based multiplexing system was designed to enable simultaneous targeting of hundreds of loci in a tissue-specific manner. Although in vivo validation of this system is ongoing, the design establishes a framework for scalable, cell-type-specific genome editing. Together, this work provides a robust platform for CRISPR-based functional genomics in zebrafish. By integrating targeted genome editing with sequencing-based validation, this approach enables systematic investigation of genes involved in cardiac development and offers a foundation for studying the genetic basis of complex diseases such as CHDs.
Recommended Citation
Argote de la Torre, Maria Fernanda, "Developing a CRISPR-Based Screen to Identify Genes Involved in Zebrafish Cardiac Development" (2026). Honors Theses. 3578.
https://egrove.olemiss.edu/hon_thesis/3578