The dynamics functions and evolution of a miniature RNA-guided enzyme

The goal of this proposal is to dissect the dynamics functions and evolution of TnpB a transposon-encodedRNA-guided endonuclease to enhance its genome-editing efficiency and expand its functions beyond DNAcleavage. While RNA-guided enzymes like CRISPR-Cas9 and Cas12 have revolutionized programmablegenome engineering there is a growing demand for smaller and more versatile enzymes. With its compactsize (~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 todevelop new RNA-guided tools with broader applications. However key aspects of TnpBs mechanism remain unclear limiting its potential. Specifically threemajor knowledge gaps need to be addressed: (1) What molecular determinants drive TnpBs editing efficiencyand how can they guide the engineering of more efficient variants? (2) How do TnpB and reRNA its guideRNA co-evolve and can this knowledge be used to design novel protein-RNA complexes? (3) How is maturereRNA generated and can this knowledge inform the design of RNA motifs with improved processingefficiency? This proposal will address these questions through three specific aims: Aim 1 will develop aquantitative kinetic model for TnpBs targeting efficiency to determine rate-limiting steps using biochemical andsingle-molecule biophysical measurements. Aim 2 will explore the co-evolution between TnpB and reRNAthrough bioinformatics structural biology and functional assays to understand how their 3D structures haveadapted together and to identify key hotspots for structural changes. Aim 3 will investigate the reRNAmaturation pathway using RNA biochemistry and structural probing focusing on how reRNA is processed intoa 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 gainingexpertise in bioinformatics for evolutionary analysis and structural biology techniques (cryo-EM RNA structuralprobing) in preparation for Aims 2 and 3. Additionally I will focus on developing essential professional skills formy independent career such as lab management mentorship and networking. This comprehensive scientificand professional training in the K99 phase will prepare me for the R00 independent phase where I will lead thecompletion of Aims 2 and 3 and establish my own research laboratory. These combined efforts will providemore quantitative and detailed mechanistic insights into TnpBs function and evolution enabling thedevelopment of future RNA-guided genome-editing tools combining compact sizes robust activities andexpanded capabilities for both research and therapeutic applications.