(295f) CRISPR/Cas-Directed Hierarchical Assembly of Protein-DNA Hybrid Nanostructures

Nanomaterials that incorporate both DNA and proteins are of particular interest in the field of bionanotechnology since they combine the highly precise, programmable assembly characteristics of Watson-Crick pairing with the chemical heterogeneity and bioactivity of proteins.¹ These hybrid structures are integral to the development of new methods of drug delivery, biosensing, and the study of fundamental characteristics and behaviors of biomolecules. Specifically, the 3D spatial control of DNA origami scaffolds enables the construction of complex shapes such as nanocages or nanotweezers. Current methods of interfacing proteins and DNA have focused on direct covalent modification of the protein of interest or fusion to independent DNA-binding protein motifs. However, the former is limited by the need for multiple reaction steps which may damage the protein, and the latter is limited by the need to change the primary amino acid sequence to recognize each distinct, corresponding DNA binding site.² A new alternative interfacing technique utilizes CRISPR-associated (Cas) nucleases, a novel class of DNA-binding proteins that possess high binding affinity (K_D ~0.05-0.5nM) and versatile binding specificity. Recent work by Lim et. al. exploits these characteristics for the spatial organization of multi-enzyme complexes on a linear double stranded DNA scaffold.³

Here, we employ a strategy that builds upon prior CRISPR/Cas-mediated assembly techniques to connect multiple distinct protein nanofibers to 3D DNA origami templates in a modular and programmable way. The protein nanofibers are comprised of -prefoldin (PFD) oligomers derived from the hyperthermophile Methanocaldococcus jannaschii, exhibit remarkable thermostability (T_M of 93 ^oC), and have been shown to be excellent scaffolds for the immobilization of enzymes and metalloproteins to enhance catalytic activity and generate conductive nanowires.^3,4 We utilize the SpyCatcher-SpyTag conjugation system to connect these fibers to a catalytically inactive variant of Cas9 nuclease derived from Streptococcus pyogenes (dCas9) that can recognize and bind to a target DNA sequence without generating a double stranded break. To accomplish this, the engineered capping protein TERM (a mutant of PFD) is genetically fused to the SpyCatcher domain (TERM-SpyCatcher) while the 13-amino acid SpyTag peptide is expressed at the C-terminus of dCas9 (dCas9-SpyTag). When combined, TERM-SpyCatcher associates selectively with the terminus of PFD filaments. Subsequent co-incubation of this product with dCas9-SpyTag generates dCas9-capped nanofibers (dCas9-fibers). For the 3D DNA origami structures (cuboids, triangles, etc.), we incorporate one to four modified staple strands which have an additional sequence of 53 base pairs extending perpendicular to each face that function as attachment points. Through the addition of dCas9-fibers to one of four distinct, corresponding sequences of single-stranded guide RNA (sgRNA) we are able to control where on the structure the fibers attach. We propose this hierarchical assembly approach can be used to generate hybrid protein-DNA nanostructures that can be further functionalized with distinct recognition moieties (e.g. enzymes, peptides, nanoparticles), and examples of such systems will be discussed.