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.

Figure 1:

Cellular uptake/biocompatibility assay of Cas9 ProSNA RNPs. (a) Flow cytometry analysis of AF647-labeled Cas9 ProSNA RNPs and Cas9 RNPs that are uptaken by HaCaT cells over 4 h. ****, p < 0.0001. (b) Cell Counting Kit-8 assay quantifying the number of viable cells upon incubation with Cas9 ProSNA RNPs. The error bars represent standard error of the mean with three independent replicates.

Figure 2:

Endosomal escape and nuclear import of Cas9 ProSNA RNPs. (a) Confocal images of HaCaT cells after incubation with 20 nM Cas9 ProSNA RNPs (red), showing endosomes (green) and the cell nuclei (blue) at various time points: 2 h, 4 h, and 8 h. Yellow regions represent colocalization between Cas9 ProSNA RNPs (red) and endosomes (green). Pink regions represent colocalization between Cas9 ProSNA RNPs (red) and the cell nuclei (blue). (b) Mander’s overlap coefficients of Cas9 ProSNA RNPs and the endosomes. (c) Nuclear import efficiency determined by the colocalization of Cas9 ProSNA RNPs within the nuclei. ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001. The error bars represent standard error of the mean with three independent replicates.

Figure 3:

Genome-editing activity of Cas9 ProSNA RNPs in mammalian cells. (a) Surveyor assays in HaCaT and hBMSCs cells. (b) Surveyor assays in RAW 264.7 cells treated with either Cas9 ProSNA RNPs or Cas9 RNPs transfected by commercial Lipofectamine CRISPRMAX^™ agent. (c) Surveyor assays of HEK293T/EGFP cells treated with Cas9 ProSNA RNPs targeting the EGFP site. The cleavage products are labeled with asterisks. (d) Flow cytometry histogram of HEK293T/EGFP cells treated with Cas9 ProSNA RNPs.

Scheme 1.

Chemical design of Cas9 ProSNAs and Cas9 ProSNA RNPs that enable delivery of Cas9 protein from the extracellular medium to the nucleus where it engages in genome editing. (I) Interaction with scavenger receptors on the cell surface and delivery via an endosomal pathway. (II) Disruption of endosomal membranes by GALA peptides. (III) Release of Cas9 ProSNA RNPs from the endosome. (IV) Nuclear import directed by nuclear localization signals (NLSs) of Cas9 ProSNA RNPs. (V) Targeting of genomic DNA and introduction of double-strand breaks.