CRISPR Spherical Nucleic Acids

• Published in: Journal of the American Chemical Society Vol. 144; no. 41; pp. 18756 - 18760
• Main Authors: Huang, Chi, Han, Zhenyu, Evangelopoulos, Michael, Mirkin, Chad A.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.10.2022
• Subjects: CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Nuclear Localization Signals - genetics; Nuclear Localization Signals - metabolism; Nucleic Acids; Peptide Hydrolases - metabolism; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.2c07913

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.