Gene editing with CRISPR technology has pioneered a new generation of possibilities regarding gene knock-in and knock-out. The Cas9 protein has been heavily utilized for many of the procedures regarding CRISPR technology, however, because of the off-target cleavages that frequently come with using Cas9 has resulted in ethical concerns. The Cas12a protein is an alternative to Cas9 and both rely on themselves to carry out editing rather than binding to other enzymes in a multi-complex structure. The increased popularity in Cas12a has come from its ability to perform multiplex genome editing and its very low percentage of off-target cleavages. However, Cas12a fails to effectively cleave to the target site once it has approached it. Previous studies have shown that modifications at the 3’ region have increased the protein cleaving ability and other studies have shown that editing the mRNA has also increased its cleaving ability. In this study four different groups of arabidopsis are compared to each other, each carrying a Cas gene in an attempt to show the benefits of using Cas12a over Cas9 in gene editing but specifically in plants as Cas12a has not been explored enough in animal cells.

Coccidioides species (C. immitis and C. posadasii) are responsible for causing coccidioidomycosis or valley fever, a fungal infection that spreads through inhalation of fungal spores. Valley fever currently affects 12 states on the West Coast of the United States and by 2095 much of the West Coast is projected to be considered endemic. While the majority of infections resolve spontaneously with minor respiratory illnesses, occasionally the infection can spread through the bloodstream from the lungs, leading to progressive, prolonged, and potentially life-threatening complications. This calls for an urgent need for the identification and characterization of novel inhibitors that can target key proteins that are essential for virulence and cellular function in C. posadasii. Interestingly, mutagenesis studies in fungal maize pathogen C. heterostrophus led to the identification of CPS1, a transmembrane protein having putative roles in key cellular mechanisms. Importantly, CPS1 mutant C. posadasii strain failed to cause the disease in healthy and immunocompromised mouse models further indicating towards the crucial role of CPS1 in disease progression. The present study aims at the identification and in-silico characterization of novel inhibitors that could target and inhibit the functions of CPS1 using a combination of molecular modeling, ligand database screening, computational docking, and molecular dynamics simulations. The best hits coming out of the computational analysis will hold tremendous potential in inhibiting the functions of CPS1 in-vitro and can be further tested in-vivo on mouse models for further validation.

The TAM family of receptor tyrosine kinases, comprising Tyro3, Axl and Mer, are important in immune signaling and cancer, but are relatively understudied. In order to develop tools for investigating their activities and roles, this research seeks to identify substrates that could be used to assay and screen inhibitors. Prior work in the Parker Lab determined substrate recognition preferences for TAM family kinases using in vitro phosphoproteomics and established computational approaches for the prediction of productive substrate-kinase interactions with up to 91% accuracy. In this current project, we have developed a computational AI-based modeling approach for designing optimal TAM family kinase substrates and have used this workflow to predict several potential TAM family peptide substrates. To test these predictions, we synthesized each peptide and employed LC-MS detection to evaluate their phosphorylation by each TAM family member. Currently, we are characterizing the Michaelis-Menten kinetics of these substrates to further understand the catalytic properties between the TAM family kinases and their predicted substrates. Identifying peptide substrates that corroborate optimal activity and selectivity will be valuable for future kinase substrate development and optimization for the TAM family, but also for other kinases that are of interest for biological characterization and drug development.

Cyclin Dependent Kinase 5 (CDK5) is a proline-directed serine/threonine protein kinase that is activated by its non-cyclin binding partners p35 and p25. The physiological activation and localization of CDK5 is guided by p35, a membrane-anchored protein. Activation by p35 is required for neuronal development and differentiation. The interaction of p25, a cleavage product p35, results in the pathological hyperactivation of CDK5 which is involved in the progression of Alzheimer's disease. To date, no major topological differences in the CDK5 binding of p35 and p25 have been reported; inhibitors which specifically block the binding of CDK5 by p25 and not p35 are hindered by these structural uncertainties. This research explores the binding potential of Pyrococcus furiosus thioredoxin (PfTrx), as a peptide aptamer scaffold, to p25 via phage display to screen for potential inhibitors of aberrant CDK5/p25 activity. The stable construct of PfTrx tolerates peptide insertions within its active site loop (-CGPC-), generating a disulfide-bridged constrained loop scaffold that serves as a new binding surface. Degenerate oligos were used in Kunkel mutagenesis reactions to insert 6-8 residues, replacing the glycine-proline stretch of the active-site loop. The mutagenized libraries are displayed on coat-protein III of M13 bacteriophage. The generated libraries will undergo plate-based affinity selections to screen for p25-specific binders. The phage derived binders of p25 will be assayed for inhibition of CDK5 following selections.