The dynamics, functions and evolution of a miniature RNA-guided enzyme - Project Summary/Abstract The goal of this proposal is to dissect the dynamics, functions, and evolution of TnpB, a transposon-encoded RNA-guided endonuclease, to enhance its genome-editing efficiency and expand its functions beyond DNA cleavage. While RNA-guided enzymes like CRISPR-Cas9 and Cas12 have revolutionized programmable genome engineering, there is a growing demand for smaller and more versatile enzymes. With its compact size (~400 amino acids), TnpB is well-suited for delivery systems with strict cargo limits, such as adeno- associated viruses (AAV). Moreover, its greater abundance and evolutionary diversity present opportunities to develop new RNA-guided tools with broader applications. However, key aspects of TnpB’s mechanism remain unclear, limiting its potential. Specifically, three major knowledge gaps need to be addressed: (1) What molecular determinants drive TnpB’s editing efficiency, and how can they guide the engineering of more efficient variants? (2) How do TnpB and reRNA, its guide RNA, co-evolve, and can this knowledge be used to design novel protein-RNA complexes? (3) How is mature reRNA generated, and can this knowledge inform the design of RNA motifs with improved processing efficiency? This proposal will address these questions through three specific aims: Aim 1 will develop a quantitative kinetic model for TnpB’s targeting efficiency to determine rate-limiting steps, using biochemical and single-molecule biophysical measurements. Aim 2 will explore the co-evolution between TnpB and reRNA, through bioinformatics, structural biology, and functional assays, to understand how their 3D structures have adapted together and to identify key hotspots for structural changes. Aim 3 will investigate the reRNA maturation pathway using RNA biochemistry and structural probing, focusing on how reRNA is processed into a functional guide RNA, and seek to design RNA motifs for more efficient reRNA processing. In the K99 mentored phase mentored by Dr. Jennifer Doudna, I will receive hands-on training in single- molecule biophysics (rotor bead tracking) with Dr. Zev Bryant (Stanford) to complete Aim 1, while also gaining expertise in bioinformatics for evolutionary analysis and structural biology techniques (cryo-EM, RNA structural probing) in preparation for Aims 2 and 3. Additionally, I will focus on developing essential professional skills for my independent career, such as lab management, mentorship and networking. This comprehensive scientific and professional training in the K99 phase will prepare me for the R00 independent phase, where I will lead the completion of Aims 2 and 3 and establish my own research laboratory. These combined efforts will provide more quantitative and detailed mechanistic insights into TnpB’s function and evolution, enabling the development of future RNA-guided genome-editing tools combining compact sizes, robust activities, and expanded capabilities for both research and therapeutic applications.
