(295f) CRISPR/Cas-Directed Hierarchical Assembly of Protein-DNA Hybrid Nanostructures
AIChE Annual Meeting
2021
2021 Annual Meeting
Nanoscale Science and Engineering Forum
Biomimetic Structures and Biomolecular Self-Assembly
Tuesday, November 9, 2021 - 2:02pm to 2:25pm
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 (TM 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.