(2il) Deep Learning-Enabled Design of Protein-Nucleic Acid Assemblies for Gene Regulation and Gene Therapy

The research goals of my lab will be focused on two thrusts: (1) deep learning (DL)-enabled computational design of protein-nucleic acid systems for applications in biotechnology; and (2) understanding the biophysics of protein-nucleic acid interactions to improve computational design tools. Protein-nucleic acid interactions are fundamental to numerous important challenges in biotechnology, including gene regulation, genome editing, therapeutics and diagnostics. As such, extensive efforts have been made over decades to develop programmable DNA or RNA binding systems that target specific sequences, notably Cys2His2 zinc finger (ZF) domains (1-2), transcription activator-like effectors (TALEs) (3-4), and CRISPR-Cas (5). Each of these tools have intrinsic limitations and leave gaps in the repertoire of available tools: ZFs can be laborious to engineer, and the size of TALE and CRISPR-Cas systems complicates their delivery in therapeutic applications. While these systems will undoubtedly continue to be improved, they have fixed overall structures which can constrain close integration with effector domains. Nature, in contrast, employs a wide diversity of binding domains which are often structurally coupled to each other and to effector regions conferring enzymatic, binding and regulatory functions (6-7). Furthermore, natural nucleic acid binding proteins are capable of recognizing and inducing formation of specific structures within the fluctuating 3D conformational landscape of nucleic acid molecules; yet programmable tools with similar function do not yet exist. Broadly, the vision of my lab will be to leverage recent advances in DL-enabled computational protein design and structure prediction for the streamlined design of custom protein scaffolds that bind specific nucleic acid sequences and structures with structurally-integrated enzymatic and regulatory functions. I expect to apply these proteins in biotechnology applications, including novel gene editing reagents and regulatory systems for use in biomanufacturing and cell-based therapeutics.

My training as a graduate student (Julius Lucks/Matthew DeLisa, Cornell University) and postdoc (David Baker, University of Washington) uniquely position me to lead this proposed research program. During my graduate studies, I designed and utilized RNA structure-based switches for regulating gene expression in bacteria (8) and applied these tools to optimize metabolic pathways for industrial biosynthesis of therapeutic proteins, biofuels, and medicinal compounds (9-10). Consequently, I developed a foundation in synthetic biology as well as molecular biology and nucleic acid biophysics. During my postdoc, I led a team to develop the first general computational method for design of sequence-specific DNA binding proteins. In this work, I utilized a variety of approaches, including state-of-the-art methods in DL-enabled and traditional physics-based protein design tools in conjunction with experimental techniques for high-through screening and biophysical characterization of designs.

Teaching Interests:

I aim to foster undergraduate education in chemical engineering through a wide variety of teaching and research activities. Specifically, I am interested in teaching core classes in the chemical engineering curriculum as well as specialty topics in synthetic biology, biomolecular engineering, and protein design. During my time as a postdoc in David Baker’s lab, I led a small group of postdocs and graduate students in teaching an NSF-funded year-long course that we designed to introduce undergraduate students to protein design research and provide the foundational knowledge to pursue individually-mentored projects. Many of the students who participated are now involved in long term research projects within the Institute for Protein Design. As a new faculty, I plan to expand on this experience to deliver biomolecular engineering courses at both the undergraduate and graduate levels, integrating the broad synthetic biology foundation from my graduate studies with the protein design specialization of my postdoctoral work. My aim will be to simultaneously provide context of the societal uses of biomolecular design, a foundational understanding of methods and approaches, as well as hands-on experience. My teaching philosophy is rooted in fostering active learning, promoting critical thinking, and encouraging collaboration. I believe that students learn best when they actively engage with the subject matter, applying theoretical concepts to practical scenarios. In addition, I have found that students are most motivated when they are provided context as to how specialized methods and technologies relate to real-world problems. By promoting critical thinking through open-ended design challenges, I will aim to cultivate students' analytical abilities, enabling them to tackle complex problems with innovative solutions. Furthermore, I encourage interdisciplinary collaboration to expose students to diverse approaches and the power of teamwork.

References:

1. Wolfe, S. A., Nekludova, L. & Pabo, C. O. DNA Recognition by Cys2His2 Zinc Finger Proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000).
2. Klug, A. The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation. Annu. Rev. Biochem. 79, 213–231 (2010).
3. Kabadi, A. M. & Gersbach, C. A. Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression. Methods 69, 188–197 (2014).
4. Joung, J. K. & Sander, J. D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).
5. Wang, J. Y. & Doudna, J. A. CRISPR technology: A decade of genome editing is only the beginning. Science 379, eadd8643 (2023).
6. Villegas Kcam, M. C., Tsong, A. J. & Chappell, J. Rational engineering of a modular bacterial CRISPR–Cas activation platform with expanded target range. Nucleic Acids Res. 49, 4793–4802 (2021).
7. Wilken, M. S. et al. Quantitative dialing of gene expression via precision targeting of KRAB repressor. bioRxiv, doi/10.1101/2020.02.19.956730 (2020) doi:10.1101/2020.02.19.956730.
8. Carlson, P., Glasscock, C., Lucks, J.B., De novo design of translational RNA repressors. bioRxiv, doi/10.1101/501767 (2018). doi.org/10.1101/501767.
9. Glasscock et al. A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation. Metabolic Engineering, 47, 488-495 (2018).
10. Glasscock et al. Dynamic control of gene expression with riboregulated switchable feedback promoters. ACS Synthetic Biology, 10, 1199-1213 (2021).