The use of bioinformatics and current genome editing tools in investigating zebrafish RB1-deficient brain tumors

This dissertation focuses on the analysis of RNA-Seq data in the context of cancer, ultimately leading to the identification of candidate genes to target for downstream analysis of their role in cell transformation and oncogenesis. Chapter 1 is a review of the current research being done with retinoblastoma deficient cancer, and the current bioinformatics solutions available for the analysis of high throughput sequence data, which will aid in molecular classification of tumor entities and in the identification of candidate genes to target for downstream analysis. Chapter 2 describes transcriptome analysis of a zebrafish RB1-primitive neuroectodermal tumor model as well as a zebrafish RB1 homozygous mutant. It begins with an introduction to the experimental design and background on both the tumor model and RB1 homozygous mutant. It then goes into the data analysis and biological interpretation of the results, from alignment of the raw reads to pathway analysis and identification of candidate genes that drive tumorigenesis. Chapter 3 of the dissertation is attributed to a candidate gene discovered in the transcriptome analysis done in chapter 2, Chromodomain-helicase-DNA-binding protein 7 (CHD7). It focuses on generating a somatic phenotype and ultimately the generation of germline mutants for genetic analysis into the role of CHD7 in normal brain development. Chapter 4 is independent of the previous two chapters, and describes the development of software to map Sleeping Beauty Transposon integration sites in the zebrafish genome. It starts off with an introduction to the Sleeping Beauty Transposon, then focuses on the filtering of the raw read data generated from the sequencing of transposon junction fragments and the pipeline that maps the transposon insertion sites to the genome, ending with molecular verification of an insertion site via PCR and sequencing. The Appendix contains all supplementary information for Chapters 3-4

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems

The use of bioinformatics and current genome editing tools in investigating zebrafish RB1-deficient brain tumors

This dissertation focuses on the analysis of RNA-Seq data in the context of cancer, ultimately leading to the identification of candidate genes to target for downstream analysis of their role in cell transformation and oncogenesis. Chapter 1 is a review of the current research being done with retinoblastoma deficient cancer, and the current bioinformatics solutions available for the analysis of high throughput sequence data, which will aid in molecular classification of tumor entities and in the identification of candidate genes to target for downstream analysis. Chapter 2 describes transcriptome analysis of a zebrafish RB1-primitive neuroectodermal tumor model as well as a zebrafish RB1 homozygous mutant. It begins with an introduction to the experimental design and background on both the tumor model and RB1 homozygous mutant. It then goes into the data analysis and biological interpretation of the results, from alignment of the raw reads to pathway analysis and identification of candidate genes that drive tumorigenesis. Chapter 3 of the dissertation is attributed to a candidate gene discovered in the transcriptome analysis done in chapter 2, Chromodomain-helicase-DNA-binding protein 7 (CHD7). It focuses on generating a somatic phenotype and ultimately the generation of germline mutants for genetic analysis into the role of CHD7 in normal brain development. Chapter 4 is independent of the previous two chapters, and describes the development of software to map Sleeping Beauty Transposon integration sites in the zebrafish genome. It starts off with an introduction to the Sleeping Beauty Transposon, then focuses on the filtering of the raw read data generated from the sequencing of transposon junction fragments and the pipeline that maps the transposon insertion sites to the genome, ending with molecular verification of an insertion site via PCR and sequencing. The Appendix contains all supplementary information for Chapters 3-4.</p

Unraveling the genomic landscape of ancestry and disease with gene expression data

The field of disease genomics aims to better understand the molecular mechanisms that underlie a disease and allow it to propagate. Additionally, is it well known that diseases disproportionally affect different populations around the world. This disproportionality is the result of many genetic and socioenvironmental factors that influence any given individual. Everyone’s genome can be thought of as a unique genomic landscape made up of many SNPs and indels that they share with their ancestry. Understanding this genomic landscape and how it affects disease prognosis and response to treatment is the goal of personalized medicine. Thanks to the many studies carried out to better understand COVID-19, cancer, and other diseases, hundreds of terabytes of RNA-Seq data is available to the public. However, much of this data does not report on a study participants’ ancestry and if so, it is often vague (Black, White, Asian) and is up to the discretion of the individual conducting the research or study participant, which allows for the possibility of human error. This dissertation introduces a tool, RNA-Seq inferred ancestry and disease (RIAD), which can infer ancestry for the 5 superpopulations; African, East/South Asian, European, and American, to a high degree of accuracy. Furthermore, RIAD has the ability to call germline and somatic mutations using solely RNA-Seq data and can infer ancestry from genomic data. In addition to unraveling the complex genomic landscapes of individuals, this dissertation presents statistical methods for better identifying cancer driver mutations in the overwhelming presence of passenger mutations that have no effect on the cancer. Lastly, the SARS-CoV2 orphan gene, ORF10, is analyzed using state of the art 3D protein structure prediction software along with correlating ORF10 variants with clinical severity using over 210K ORF10 sequences from a clinical dataset

Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation

In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4(rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells.This article is published as Schultz, Laura E., Jeffrey A. Haltom, Maira P. Almeida, Wesley A. Wierson, Staci L. Solin, Trevor J. Weiss, Jordan A. Helmer, Elizabeth J. Sandquist, Heather R. Shive, and Maura McGrail. "Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation." Disease models & mechanisms 11, no. 6 (2018): dmm034124. doi: 10.1242/dmm.034124.</p

Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation

In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells. This article has an associated First Person interview with the first author of the paper

Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.This article is published as Wierson, Wesley A., Jordan M. Welker, Maira P. Almeida, Carla M. Mann, Dennis A. Webster, Melanie E. Torrie, Trevor J. Weiss et al. "Efficient targeted integration directed by short homology in zebrafish and mammalian cells." Elife 9 (2020): e53968. doi: 10.7554/eLife.53968.</p

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems.This is a pre-print made available through bioRxiv, doi: 10.1101/431627.</p