A protein design strategy is used that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs, establishing a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
Abstract
The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome-editing capabilities. However, generating diverse multidomain proteins with robust enzymatic properties remains challenging. Here, we use a protein design strategy that couples a structure-guided inverse-folding model with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease, termed SynTnpBs. High-throughput screening of artificial intelligence-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant, and human cells. Cryo-electron microscopy-based structure determination of the most divergent variant revealed stabilizing contacts in the RNA-DNA interfaces across conformations, demonstrating the design potential of this approach. Together, these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space.
A PLM-assisted physics-driven approach is presented that utilizes atomistic molecular dynamics simulations and automated path searching to efficiently obtain the complete kinetic insights, including the transition state structures, for the conformational changes of Cas before DNA cleavage.
Natural variation in PAM recognition among SaCas9 orthologs is analyzed and StaCas9 is identified as a compact and efficient nuclease recognizing an NNG PAM, establishing StaCas9 as a high-performance genome-editing tool for therapeutic applications.
Using high-throughput junction mapping together with large-scale comparative genomics, this work redefined the in vivo structural boundaries, growth, and mobilization of IS110 elements and uncovered a previously unrecognized size continuum extending to ∼100 kb, driven by progressive additions.
Kuang Hu, Bingliang Xie, HengYi Yang et al.· bioRxiv· 0 citations
The rapid advancement of protein engineering and genetic code expansion technologies over the last decade has reshaped how researchers rationally design proteins with novel catalytic functions. Among these approaches, the site-specific incorporation of unnatural amino acids has enabled the introduction of chemical functionalities that are inaccessible to the canonical amino acid space. In this perspective, we highlight the metal-chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) and its emerging utility in mediating nucleic acid cleavage. Multiple studies have demonstrated the successful site-specific incorporation of BpyAla into proteins of interest, where subsequent metal coordination enables catalytic cleavage of DNA and RNA substrates. Here, we discuss the potential of BpyAla-mediated nucleic acid cleavage, with emphasis on the development of next-generation BpyAla analogues, the exploration of alternative metal cofactors, cooperative and multi-residue design strategies, and the expansion of compatible protein scaffolds and nucleic acid substrates. Designable BpyAla-engineered systems represent an emerging frontier in artificial metallonuclease design, with potential long-term relevance to targeted nucleic acid therapeutics.
E. Lundrigan, Matthew T. O’Neill, J. P. Pezacki· Frontiers in Chemical Biolog...· 0 citations
An RNA-guided bridge recombinase system is engineered through rational mutagenesis and AI-assisted directed evolution, enabling programmable chromosomal rearrangements in both plant and mammalian cells and achieving up to a 29.8-fold increase in activity.
Rui Gao, Jingjing Wei, Chao Sun et al.· Trends in Biotechnology· 0 citations
