DNA-Binding Proteins (DBPs) hold a strong affinity to interact with the major grooves of DNA for the purposes of transcription, translation, and repair. Although DBPs are found in nature, their specificity is difficult to predict and their production expends excessive resources. Therefore, our project’s goal is to efficiently generate DBPs that allow us to enable exact processes to occur. This is promising for future use in treating genetic diseases. In our research, we studied point mutations in the hexosaminidase subunit alpha (HEXA) gene and adenosine deaminase (ADA) gene, which can lead to Tay-Sachs disease (TSD) and Severe Combined Immunodeficiency (SCID), respectively. TSD is a rare genetic disease that affects infants and ultimately leads to brain damage, resulting in these children not making it to grade school age. Similarly, SCID is genetic, where children lack a strong immune system. This increases their susceptibility to infections, especially during their first year of life. Targeting the HEXA and ADA genes, we first developed designs utilizing computational software like RosettaFold, RFdiffusion, x3DNA, and LigandMPNN, followed by rigorous filtering via RosettaFold Nucleic Acid. Afterwards, we tested the final designs using yeast cultures and fluorescence-activated cell sorting (FACS) in the laboratory to determine which bind best to their generated DNA template sequences. Overall, we expect to find a few specific DBPs that bind effectively as predicted during the computational pipeline. These successful designs can be utilized as genome-editing proteins, correcting their target DNA sequences and restoring normal function.

Climate change and global population growth are driving the need for more sustainable methods for growing crops used in agriculture and the production of biofuels. To address these challenges we are exploring the role of the plant microbiome in host plant tolerance to environmental stresses related to climate change. In particular, we are investigating whether endophytes, microorganisms that colonize the internal tissues of plants, make the host plants more tolerant to drought or low-nitrogen conditions. We are currently optimizing the process of DNA extractions of fruit and poplar trees which were inoculated with beneficial nitrogen-fixing bacteria, and grown in either water or nitrogen-limited conditions. We then purify high quality microbial DNA and use polymerase-chain reaction (PCR) to optimize strain-specific primers (SSP), which target specific DNA sequences in the genomes of our endophytes in the presence of competing DNA. This allows us to gain an understanding of where they colonize, and to demonstrate that the trees were successfully colonized by our endophytes to support growth and drought tolerance data collected from inoculated and uninoculated controls. By ensuring the SSPs only target our strains of interest, we differentiate our endophytes from other members of the plant microbiome. These primers are then used in Droplet Digital PCR (ddPCR) to quantify their relative abundance. Using this information we hope to demonstrate that beneficial endophytes can be used as a sustainable method for improving drought and low nitrogen tolerance in plants, both in agricultural and biofuel applications, reducing the consumption of nitrogen fertilizers and water for irrigation in these sectors.

Human immunodeficiency virus type 2 (HIV-2) is endemic in West Africa and is also found in areas with socioeconomic ties to the region. Historically, advancements in the treatment of HIV-2 infection have been slow compared to HIV-1, and there is an urgent need to identify effective antiretrovirals (ARVs) for people with HIV-2 (PWH2) who harbor drug-resistant virus. Lenacapavir (SUNLENCA®) may help fill this gap in the HIV-2 treatment landscape. Lenacapavir is a long-acting, injectable ARV that targets the HIV-1 capsid protein and is approved for use in adults with multidrug-resistant HIV-1 infection. We recently showed that lenacapavir is highly active against HIV-2 isolates in culture, albeit with a 11- to 14-fold lower potency relative to HIV-1. These data suggest that lenacapavir binds to HIV-2 capsid cores in a manner similar to that observed in HIV-1. To further test this hypothesis, I am using site-directed mutagenesis (SDM) to introduce specific amino acid changes into the putative lenacapavir-binding region of the HIV-2 capsid protein. These changes fall into two categories: 1) mutations that convert the targeted residue in HIV-2 to the corresponding amino acid residue found in HIV-1; and 2) mutations that are associated with the development of lenacapavir resistance in patients with HIV-1 infection. To date, I have constructed a 5.5-kilobase plasmid clone that contains the capsid-encoding region of HIV-2ST (plus downstream flanking sequences) in a pBluescript plasmid vector. I introduced the desired mutations by SDM, and verified that the plasmids are correct via automated Sanger sequencing. My next step is to clone the mutated capsid sequences into a full-length clone of the HIV-2 ST genome for virus production and culture-based drug susceptibility testing. These studies have direct implications for improving HIV-related care and public health in West Africa and other areas where significant numbers of PWH2 reside.

The endangered Western Pond Turtle (Actinemys marmarata) was once a broadly distributed species across a large range from Western Washington to as far south as Baja, Mexico. However, unprecedented amounts of population loss driven by exploitation as a food source, wetland development and the destruction of habitat began to plague this species beginning in the early 1900s. As a result, the Washington State Department of Fish and Wildlife (WDFW) and the Woodland Park Zoo created six reestablishment sites that proved to be successful, elevating the number of turtles from 150 in the 1990s to around 900 individuals today. Threats to this species persist, making it more crucial than ever to locate new reintroduction sites to increase population numbers and promote self-sustaining populations. Our analysis is focusing on determining if the Union Bay Natural Area (UBNA) could be a potential 7th reestablishment site through the utilization of a habitat suitability index (HSI) with ArcGIS Pro and comparisons with Klickitat County and Pierce County sites. We anticipate that UBNA displays the habitat characteristics capable of promoting occupancy by the Western Pond Turtle. Furthermore, given UBNA’s popularity as a bird watching hotspot, we will also establish a kiosk intended to raise public awareness, educate the public about its threats, and display a map demonstrating their current and potential re-establishment zones as shown by our analysis. This kiosk includes a website linking the general public to a survey allowing visitors to record turtle sightings that can be accessed by future researchers. This study has widespread implications in terms of wildlife conservation, endangered species recovery and the management of threatened species.

Orofacial cleft (OFC) is a relatively common birth defect that has major impacts on affected individuals and their families. Underlying causes of OFC include genetics and environmental influences. Genome-wide association studies (GWAS) and linkage analyses have revealed genes in which DNA variants are enriched in OFC cases relative to in unaffected individuals in the same ethnic group. Only a portion of the genetic causes have been identified. Here we focus on ARHGAP29, which was identified in several GWAS of OFC. To uncover the role of this gene in craniofacial morphogenesis, and to identify other members of its regulatory pathway, we are working on deleting a paralog of this gene, arhgap29b, in zebrafish embryos. To this end, we have designed four CRISPR guide RNAs that target specific exons in the arhgap29b gene and injected them into zebrafish embryos. We predict that such embryos will a) harbor mutations in the arhgap29b gene, which we plan to test with PCR and sequencing, and b) display abnormal morphogenesis of the face, which we plan to test by microscopy. Alternatively, we may observe a) but not b). In this event we would simultaneously disrupt the other paralog, arhgap29a. These findings will advance our understanding of genes associated with orofacial cleft, hopefully leading to improved diagnosis and underpinning the design of therapies for this disorder.