Research ArticleGENETICS

Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform

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Science Advances  03 Apr 2019:
Vol. 5, no. 4, eaav7199
DOI: 10.1126/sciadv.aav7199
  • Fig. 1 Design of the UCNP-based CRISPR-Cas9 delivery system for NIR light–controlled gene editing.

    (A) Preparation of UCNPs-Cas9@PEI. (B) NIR-triggered delivery of Cas9-sgRNA to the nucleus of the cell for gene editing: (I) attachment to the cell membrane, (II) endocytosis, (III) endosome escape, (IV) release from particles and step into the nucleus, and (V) search for the target DNA locus and initiate the DNA double-strand break for genome editing.

  • Fig. 2 Characterization of the nonviral nanovehicles.

    TEM images of (A) oleate-stable UCNPs, (B) UCNPs@SiO2, and (C) UCNPs-Cas9@PEI and its EDX elemental mapping. Scale bars, 50 nm. (D) Fluorescence spectrum of the UCNPs activated by a 980-nm laser (black line) and UV-vis absorption spectrum of 4-(hydroxymethyl)-3-nitrobenzoic acid (ONA) (green line). a.u., arbitrary units. (E) The ζ potentials of Cas9 protein, Cas9/sgRNA, UCNPs@SiO2, UCNPs@SiO2-COOH, UCNPs@SiO2-ONA, UCNPs-Cas9, and UCNPs-Cas9@PEI (from left to right).

  • Fig. 3 NIR light–controlled release of Cas9 for gene editing.

    (A) UV-vis absorption (280 nm) of the supernatant of UCNPs-Cas9 with different irradiation times by a 980-nm laser. (B) Schematic illustration of internalization of Cas9 that enters the nuclei and triggered by NIR light for gene editing. (C) CLSM images of cells incubated with UCNPs-Cas9@PEI for 6 hours with or without 980-nm irradiation (red, Cas9 labeled with Cy3; blue, nuclei stained with DAPI). Scale bar, 10 μm. (D) Fluorescence microscopy images of KB cells treated with different formulations (I, NIR only; II, UCNPs@PEI + NIR; III, UCNPs-Cas9@PEI without NIR; IV, UCNPs-Cas9@PEI + NIR). Green, EGFP; blue, nuclei stained with DAPI. Scale bar, 100 μm. (E) Flow cytometry (FCM) analysis of EGFP disruption in cells treated with (I) NIR only, (II) UCNPs@PEI + NIR, (III) UCNPs-Cas9@PEI without NIR, and (IV) UCNPs-Cas9@PEI + NIR. Bars represent mean ± SD (n = 5). The EGFP silencing efficiencies of UCNPs-Cas9@PEI and UCNPs-Cas9@PEI + NIR were 35.3 and 6.9%, respectively.

  • Fig. 4 NIR-mediated gene editing induced apoptosis of A549 cells.

    (A) Schematic illustration of the cancer cell killing triggered by NIR. (B) Western blot assay. (C) T7EI assay. (I) Pure media + NIR, (II) CRISPR-Cas9@PEI + NIR, (III) UCNPs@PEI + NIR, (IV) UCNPs-Cas9@PEI without NIR, and (V) UCNPs-Cas9@PEI + NIR. (D) CCK-8 analysis of A549 cells treated with pure media only, CRISPR-Cas9@PEI + NIR, UCNPs@PEI + NIR, UCNPs-Cas9@PEI without NIR, and UCNPs-Cas9@PEI + NIR (from left to right). CLMS images of cells treated by (E) pure media + NIR, (F) CRISPR-Cas9@PEI + NIR, (G) UCNPs@PEI + NIR, (H) UCNPs-Cas9@PEI without NIR, and (I) UCNPs-Cas9@PEI + NIR. Green, calcein-AM; red, PI. Scale bar, 200 μm. (J) Flow cytometry assay of cell apoptosis. The mean value was analyzed using t test (n = 5). **P < 0.01 represents significant difference and ****P < 0.0001 represents highly significant difference.

  • Fig. 5 The drug administration in a xenograft model of A549 cells.

    (A) Schematic diagram of the working mechanism of remote-controlled oncotherapy in vivo. (B) Tumor sizes after different treatments, as indicated. (C) Weights of tumors treated by (I) PBS + NIR, (II) UCNPs@PEI + NIR, (III) UCNPs@PEI + Cas9 free + NIR, (IV) UCNPs-Cas9@PEI, and (V) UCNPs-Cas9@PEI + NIR (from left to right). (D) Changes in mouse body weight after different treatments, as indicated. (E) Images of tumor with PBS + NIR (top row), UCNPs-Cas9@PEI without light (middle row), and UCNPs-Cas9@PEI + NIR (bottom row). (F) H&E staining (scale bar, 200 μm), TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining (scale bar, 100 μm), KI67 antigen staining (scale bar, 100 μm), and caspase-3 staining (scale bar, 100 μm) of tumor tissues. The mean value was analyzed using t test (n = 5). **P < 0.01 represents significant difference and ***P < 0.001 represents highly significant difference.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaav7199/DC1

    Fig. S1. Schematic for the synthesis of UCNPs-Cas9.

    Fig. S2. The structure of UCNPs confirmed by HRTEM and SAED pattern.

    Fig. S3. The surface modification process of UCNPs.

    Fig. S4. Photocleavage reaction triggered by NIR light on the surface of UCNPs.

    Fig. S5. Cellular internalization of UCNPs-Cas9@PEI monitored by confocal microscopy.

    Fig. S6. The viability of cells treated under different conditions.

    Fig. S7. The assay of EGFP expression in cells.

    Fig. S8. Cell viabilities observed by CLSM images.

    Fig. S9. The biocompatibility analysis of UCNPs-Cas9@PEI.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Schematic for the synthesis of UCNPs-Cas9.
    • Fig. S2. The structure of UCNPs confirmed by HRTEM and SAED pattern.
    • Fig. S3. The surface modification process of UCNPs.
    • Fig. S4. Photocleavage reaction triggered by NIR light on the surface of UCNPs.
    • Fig. S5. Cellular internalization of UCNPs-Cas9@PEI monitored by confocal microscopy.
    • Fig. S6. The viability of cells treated under different conditions.
    • Fig. S7. The assay of EGFP expression in cells.
    • Fig. S8. Cell viabilities observed by CLSM images.
    • Fig. S9. The biocompatibility analysis of UCNPs-Cas9@PEI.

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