Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing

Abstract

CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids.

Keywords: CRISPR-Cas9; DNA; drug delivery; genome editing; nanoparticles.

Figure 1

Schematic design of the DNA NC mediated CRISPR-Cas9 delivery system. (a) Preparation of Cas9/sgRNA/NC/PEI. I: The NC was synthesized by RCA and loaded with the Cas9/sgRNA complex through Watson-Crick base pairing; II: PEI was coated onto Cas9/sgRNA/NC for enhanced endosome escape. (b) Delivery of Cas9/sgRNA by the DNA NC based carrier to the nucleus of the cell for genome editing. I: Bind to cell membrane; II: Endocytosis; III: Endosome escape; IV; Transport into the nucleus; V: Search for target DNA locus in the chromosome and introduce double strand breaks for genome editing.

Figure 2

Particle characterization of Cas9/sgRNA/NC-12/PEI. (a) Monitoring zeta potential of the Cas9/sgRNA/NC-12/PEI assembly process. Bars represent mean ± SD (n = 3). (b) Hydrodynamic size distribution of Cas9/sgRNA/NC-12/PEI. (c) AFM image and d) TEM image of Cas9/sgRNA/NC-12/PEI with scale bars of 400 nm and 100 nm, respectively.

Figure 3

a) CLSM images of U2OS.EGFP cells incubated with Cas9/sgRNA/NC-12/PEI for 1 h, 2 h, 4 h and 6 h (Cas9 and sgRNA concentrations at 100 nM). Green for EGFP, red for Cas9 stained with AF647 and blue for nuclei stained with Hoechst 33342. Scale bar is 10 µm. b) Relative Cas9/sgRNA/NC-12/PEI uptake by U2OS.EGFP cells in the presence of different endocytosis inhibitors (Cas9 and sgRNA concentrations at 100 nM). **P<0.01 as compared to the control group. Bars represent mean ± SD (n = 3). c) In vitro cell viability of U2OS.EGFP cells treated with Cas9/sgRNA/NC-12/PEI and Cas9/sgRNA/PEI by flow cytometry. The cells were stained with TO-PRO-3 live/dead stain after the treatment and analyzed by flow cytometry. Bars represent mean ± SD (n = 3).

Figure 4

Genome editing by Cas9/sgRNA delivered by DNA NC (8 µg/mL) coated with PEI (10 µg/mL). a) Fluorescent microscope images and flow cytometry analysis of U2OS.EGFP cells treated with Cas9/sgRNA/PEI and Cas9/sgRNA/NC-12/PEI (Cas9 and sgRNA concentrations at 100 nM). Green represents EGFP and blue represents nuclei stained with Hoechst 33342. Scale bar is 100 µm. b) T7EI assay of U2OS.EGFP cells treated with Cas9/gRNA/NC-12/PEI and Cas9/gRNA/PEI. c) EGFP disruption assay of Cas9/gRNA delivered by different DNA NCs. Percentages of EGFP negative cells after treating with Cas9/sgRNA/NC-23/PEI, Cas9/sgRNA/NC-12/PEI, Cas9/sgRNA/NC-0/PEI and Cas9/sgRNA/PEI at different Cas9/sgRNA molar ratios were profiled. Bars represent mean ± SD (n = 3).

Figure 5

In vivo delivery of Cas9/sgRNA into U2OS.EGFP xenograft tumors in nude mice. Tumor sections were collected 10 days after intratumoral injection of Cas9/sgRNA/NC-12/PEI. The EGFP was stained by FITC conjugated GFP antibody and nuclei were stained with Hoechst 33342. Scale bar is 50 µm.