Joubert syndrome (JS) is a genetic neurodevelopmental disorder that affects ~1 in 100,000 live births. JS is diagnosed by a distinctive hindbrain malformation that manifests as the “molar tooth sign” on axial brain imaging. Remarkably, >40 genes have been associated with JS, making it one of the most genetically heterogeneous Mendelian conditions. The clinical and brain imaging features of people with JS display a broad range of severity. In fact, we have identified a substantial number of individuals without the molar tooth sign but that have imaging features suggestive of JS. It is not known whether these “JS-like” patients represent the mild end of the phenotypic spectrum associated with variants in JS genes or a different set of genetic disorders. It is also not known whether these JS-like patients are at risk for the progressive retinal, kidney and liver disease seen in some JS patients. To answer these questions, I performed targeted DNA sequencing of the JS genes in JS-like patients, and I used an in-house bioinformatics pipeline to identify predicted-pathogenic variants. We hypothesize that a large subset of JS-like patients will have genetic causes in JS genes. If this hypothesis is supported, we will expand the phenotypic spectrum associated with variants in JS genes and improve the medical care of JS-like patients by supporting monitoring of JS-associated progressive features and sequencing of JS genes in these patients. This will also be proof of concept for evaluating mild clinical presentations of other conditions to determine if they share the same genetic causes.

Ageing is intrinsic to life, and its progression is a major risk factor for many high-morbidity diseases. Through examination of the cellular processes that govern aging, we hope to gain insight into how to reduce not only the rate of aging, but the incidence of associated diseases as well. Genomic instability is one of the key hallmarks of ageing and occurs in both the mitochondrial and nuclear genomes of both humans as well as less complex invertebrate models. Furthermore, loss of mitochondrial DNA stability is also associated with a loss of nuclear genome stability. In addition to producing essential electron transport chain proteins, mitochondria also produce essential iron-sulfur cluster proteins that are necessary for repair functions within the nuclear genome. Our goal is to disentangle the connections between nuclear and mitochondrial genome degradation using fluorescent reporters in Saccharomyces cerevisiae, in conjunction with a novel microfluidic system. Nuclear DNA degradation will be measured using RAD52::GFP, a component of the DNA-damage repair pathway, while the mitochondrial response will be observed with RTG1::mCh, which signals mitochondrial dysfunction. Since RTG1 and RAD52 both localize to the nucleus during DNA damage events, the relative concentrations of these proteins and their temporal patterning will reveal which system tends to fail first. We have engineering a novel strain of Saccharomyces cerevisiae that satisfies these flourescent properties, and will use a microfluidic chip to explore the interaction between both the nuclear and mitochondrial DNA, and determine the timing and causality of the genomic feedback loop that has been previously described.

Many proteotoxic diseases, such as Alzheimer's disease, exhibit reduced or impaired mitochondrial functionality. Recent studies have shown that some classic retrograde response signals, which act to counter mitochondrial dysfunction, can mitigate the severity of some of these diseases. Our lab recently identified a novel retrograde response pathway controlled by a core mitogen-activated protein kinase cascade, composed of DLK-1, SEK-3 and PMK-3 (MAP3K/MAP2K/MAPK), that in turn signal to a collection of downstream bZIP transcription factors. In this study, we examined whether this novel retrograde response could alter the susceptibility to amyloid-beta (Aβ) aggregation and paralysis in a worm model of Alzheimer’s disease. We conducted several RNAi screens to determine the effect of removing individual bZIP transcription factors and core MAPK components on Aβ aggregation. We found that our retrograde response indeed plays an important role in countering the severity of paralysis in Aβ-producing worms, and that this effect is largely mediated by a single bZIP transcription factor. We will discuss these results and show the surprising tissues in which the retrograde response must act to exhibit its Aβ toxicity mitigating-effects in worms.

One fundamental difference between sexes is that females have two X chromosomes, and males have one. This leads to an X chromosome gene dosage imbalance between sexes. X chromosome inactivation (XCI) in females is the process where one X chromosome is inactivated to balance gene dosage. However, some genes remain expressed from the inactive X (Xi) resulting in higher gene expression in females, suggesting these genes may play a female-specific role. My project focuses on Kdm6a, an X-linked escape gene that encodes a histone demethylase that removes trimethylation on lysine 27 of histone 3 (H3K27me3), a histone modification associated with gene repression and highly enriched on the Xi. Using hybrid embryonic stem cells (ES) with a Kdm6a knock out (KO), I contributed to a study demonstrating that KDM6A enhances gene expression in a maternally biased manner, suggesting it is capable of distinguishing parental alleles of genes. I then explored whether KDM6A also regulates allelic expression from the Xi. We hypothesized that Kdm6a KO will lead to decreased escape gene expression from the Xi via increased H3K27me3 at the promoters of escape genes. We have established an F1 hybrid ES cell model to ablate KDM6A protein levels by CRISPR/Cas9. Importantly, these cells have skewed XCI, which facilitates measurements of gene regulation by KDM6A on the Xi. So far, I have shown that Kdm6a KO leads to reduced expression and complete loss of the protein. I confirmed that KO cells retain both X chromosomes in culture and that KO results in reduced capability for differentiation. Next, we initiated studies to measure allele-specific expression of X-linked genes and to determine whether gain of H3K27me3 due to loss of KDM6A may explain expression changes on the Xi. Results from this study will help identify potential therapeutic targets for individuals with super numery X chromosomes.

Non-coding regions account for 98% of the genome and harbor regulatory elements that play a significant role in regulating and maintaining levels of gene expression via transcriptional and translational control mechanisms. Our lab focuses on the functional characterization of cis-regulatory regions (CREs) in the human retina and identifying variants within these elements that contribute to inherited retinal diseases. Current research efforts often leverage high throughput functional assays to identify putative CREs at a moderate level of certainty, however, little can be reliably inferred about the causality of variants within CREs without further experimental or computational analysis. Moreover, much still remains unknown regarding the molecular interactions that give rise to the observed phenotypic abnormalities. As such, the primary aim of my capstone project is to design a bioinformatics pipeline that will synthesize multi-dimensional epigenetic sequencing data into a single quantitative metric of single-nucleotide variant (SNV) impact at a base pair resolution. For a given nucleotide, its impact score speaks to the magnitude and directionality of the predicted effect of the mutation on its contribution to CRE activity. The pipeline integrates in silico mutagenesis with machine learning approaches such as Hidden Markov Models and Support Vector Machines to derive variant impact scores within putative retinal CREs. In tandem with preliminary high throughput sequencing data on the adult human retina, this workflow will ultimately facilitate a more robust interpretation of causal non-coding mutations and aid in the evaluation of potential retinal disease modeling platforms. The resulting output can potentially drive more well-informed high confidence diagnostics.

CRISPR is a powerful gene-editing tool with several advantages over state-of-the-art viral vectors used for gene therapy. However current methods to deliver CRISPR to human cells requires electroporation, which is cytotoxic and not viable for in vivo delivery. The Adair lab previously demonstrated gold nanoparticles (AuNPs) to passively deliver CRISPR gene editing into blood stem/progenitor cells, with Cas12a (Cpf1) nuclease resulting in high levels of homology-directed repair (HDR) compared to Cas9 when co-delivered with a single-stranded, homology-directed DNA template (HDT). Here we tested this AuNP-mediated delivery system in primary human T cells. We hypothesized that AuNP delivering Cpf1+HDT would also mediate high levels of HDR in T cells, compared to Cas9+HDT. To facilitate HDR readouts, the HDT encoded an 8 bp NotI restriction enzyme site. Both guide RNAs targeted the same C-C chemokine receptor type 5 (CCR5) gene locus. A mutation at this locus confers resistance most human immunodeficiency virus (HIV) strains. Briefly, CD3+ T cells from 8 different human donors (n=8 biological replicates) were either cultured overnight and exposed to AuNP-CRISPRs, or activated with CD3/CD28 beads prior to AuNP-CRISPR treatment. Test conditions for each donor included Mock-treated cells (negative control), naked AuNP, AuNP-CRISPR/Cpf1, AuNP-CRISPR/Cpf1+HDT, AuNP-CRISPR/Cas9, or AuNP-CRISPR/Cas9+HDT at identical cell and AUNP concentrations for 48 hours. Cell viability and counting by trypan blue dye exclusion assay demonstrated >70% viability and yield across all conditions tested. Gene editing analysis by tracking of indels by decomposition (TIDE), demonstrated the highest levels of gene editing with AuNP-CRISPR/Cpf1+HDT, with >90% of all editing being successful HDR. In non-activated T cells, mean gene editing levels were 19.7% across all donors (n=4). Final results for activated T cells are currently pending. These data demonstrate AuNP-mediated CRISPR/Cpf1 delivery as an efficient method for HDR in primary human T cells and suggest utility in human clinical applications.

Congenital heart defects have been linked to numerous genes, but many of the genes responsible are not yet identified. The purpose of this research is to identify the unknown genetic causes of human congenital heart defects, utilizing zebrafish as a model organism. Using CRISPR-Cas9 to edit the genome of zebrafish, we are creating mutations in genes we predict are involved in human congenital heart defects. Our lab has used a CRISPR-based screen in zebrafish to identify new genes that, when knocked out, lead to defective heart development in zebrafish embryos. For this research project, our questions are: Can we associate specific, CRISPR-induced genetic mutations in these genes with our heart-defective zebrafish embryos? And, can we genetically engineer heritable mutations in these respective genes in zebrafish? The methods used in this project involve using several sets of DNA oligonucleotide primers to assess where and how the CRISPR reagents have altered the screened candidate genes. We have analyzed three genes—grpel1, pomp, and psmd6—each with four CRISPR target sites. The primer testing and animal genotyping have been done using PCR, gel electrophoresis, and gel imaging, with genotyping also requiring restriction digests. Our results have been promising. First, we determined which CRISPR target sites are effective for each gene. Second, we successfully identified CRISPR-induced mutations in F0-generation animals for each of these three genes. Third, for the pomp gene, we identified germline-transmission of a specific CRISPR mutation corresponding with heart-defective embryos. This result identifies pomp as a new candidate gene for heart defects. A key implication of these findings is that we can successfully create lineages of zebrafish carrying mutations in these new heart defect genes. Our work will allow for further testing and a better understanding of the genetics behind heart development.

Osteoporosis is an orthopedic disease in which old bone begins to dissolve but is not replaced by new bone. This reduces overall bone density and increases a patient’s risk for fractures. One human gene associated with osteoporosis-related traits is WNT16, which is also expressed in zebrafish. Previous studies in our lab have shown that wnt16 mutant fish have skeletal defects. The goal of this project was to find the most effective method for knocking out genes associated with osteoporosis in zebrafish using CRISPR/Cas9 technology. Once Cas9 creates a double-strand break in the DNA, there are different methods of DNA repair, two of which are non-homologous end joining (NHEJ) and microhomology mediated end joining (MMEJ). In this study, we designed guide RNAs (gRNAs) targeting wnt16 to compare a more predictable, MMEJ-based CRISPR approach to the previous, NHEJ-based CRISPR approach used in the lab. Next, we assessed gene editing in zebrafish embryos injected with two new gRNAs biased toward MMEJ repair to determine their efficacy in knocking out wnt16. Using DNA sequencing and analysis, we found that injections of both MMEJ gRNAs caused high rates of insertions and deletions (indels) compared to a control group which was not injected with gRNA. Moreover, the predicted MMEJ indels were found to be in high abundance in DNA sequences from injected fish. Further, we detected anticipated morphological differences expected from loss of wnt16 in the fish injected with MMEJ gRNAs compared to the control fish, suggesting that the MMEJ gRNAs work as expected. Based on these results, MMEJ-biased gRNA design appears to be a promising approach to improve efficiency in knocking out zebrafish genes. This project may help optimize a rapid and effective screening of many zebrafish candidate genes using CRISPR/Cas9 technology to study skeletal phenotypes in zebrafish.