CRISPR Spherical Nucleic Acids

Abstract

The use of CRISPR/Cas9 systems in genome editing has been limited by the inability to efficiently deliver the key editing components to and across tissues and cell membranes, respectively. Spherical nucleic acids (SNAs) are nanostructures that provide privileged access to both but have yet to be explored as a means of facilitating gene editing. Herein, a new class of CRISPR SNAs are designed and evaluated in the context of genome editing. Specifically, Cas9 ProSNAs comprised of Cas9 cores densely modified with DNA on their exteriors and preloaded with single-guide RNA were synthesized and evaluated for their genome editing capabilities in the context of multiple cell lines. The radial orientation of the DNA on the Cas9 protein surface enhances cellular uptake, without the need for electroporation or transfection agents. In addition, the Cas9 proteins defining the cores of the ProSNAs were fused with GALA peptides on their N-termini and nuclear localization signals on their C-termini to facilitate endosomal escape and maximize nuclear localization and editing efficiency, respectively. These constructs were stable against protease digestion under conditions that fully degrade the Cas9 protein, when not transformed into an SNA, and used to achieve genome editing efficiency between 32 and 47%. Taken together, these novel constructs and advances point toward a way of significantly broadening the scope of use and impact of CRISPR-Cas9 genome editing systems.

Inspired Beyond Nature: Three Decades of Spherical Nucleic Acids and Colloidal Crystal Engineering with DNA

“…This example represents perhaps the first clinical evidence of the importance of “structural immunotherapy.” In a related area, intracellular “nanoflare”-inspired, quencher-free detection schemes were developed based on forced intercalation and protein-based structures with increased applicability and decreased background signal and false-positives . Moreover, protein-core SNAs were utilized in therapeutic protein delivery , and to generate CRISPR SNAs aimed at expanding cell and tissue access for this important gene editing platform.…”

Endogenous Enzyme-Activatable Spherical Nucleic Acids for Spatiotemporally Controlled Signal Amplification Molecular Imaging and Combinational Tumor Therapy

“…Inspired by this, spherical nucleic acids (SNAs) constructed from nanomaterial cores, including upconversion nanoparticles (UCNPs), gold nanoparticles, and micelles with high density-oriented nucleic acid shells, have been widely developed due to their adjustable hybridization activity, controllable structure and size, antinuclease digestion stability, and superior endocytosis. , During the construction process of SNAs, click chemistry is often used as a favorable synthetic tool owing to the advantages such as simple experimental procedures, mild reaction conditions, and high selectivity. , It is worth noting that Bertozzi et al proposed a copper-free strain-promoted azide–alkyne cycloaddition click (SPAAC) reaction in 2004 . This new strategy was subsequently widely used in oligonucleotide labeling, nanomaterial functionalization, hybridization, and block copolymer formation. − …”

Construction and bioanalytical applications of poly-adenine-mediated gold nanoparticle-based spherical nucleic acids

“…Since the pioneering work of Mirkin in 1996, 1 there has been great interest in constructing SNAs with various metal-based nanoparticles, such as gold nanoparticles (AuNPs), 2,3 quantum dots, 4 iron oxide, 5,6 silver, 7,8 or metal-free nanoparticles, such as hollow silica, 9 liposomes, 10 micelles, 11 cross-linked oligodeoxyribonucleotides, 12 and proteins. 13 The wide range of SNAs that have been generated indicates that their properties are not reliant on the core but instead determined by the distinct structure of the nucleic acid shell. This sets them apart from linear nucleic acids.…”