Research ArticleMICROTECHNOLOGY

CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation

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Science Advances  14 Aug 2015:
Vol. 1, no. 7, e1500454
DOI: 10.1126/sciadv.1500454
  • Fig. 1 Delivery mechanism and device design.

    (A) Illustration of the delivery process wherein cells pass through the microconstriction and experience deformability. Plasmids encoding sgRNA and Cas9 protein are mixed with the cells to flow through the chip. (B) Illustration of the delivery mechanism whereby transient membrane holes are generated when cells pass through the microconstriction and specific genome editing is conducted after plasmids encoding sgRNA and Cas9 protein are delivered into the cell. Cell deformation was shown by microscopy when cells passed through the microconstriction. Scale bar, 15 μm. nt, nucleotide. (C) Microscopy of the whole device structure. Scale bar, 0.5 mm. Scanning electron microscopy (SEM) of scattered and deformable zones in the device is also shown. Scale bar, 15 μm. One diamonded microconstriction of 15-μm depth and 4-μm width is indicated by the red arrow. The length of the diamond edge is 10 μm. (D) Cell stress simulation was applied on the diamonded microconstriction design with 15-μm depth and 4-μm width when a cell began to penetrate the constriction. A graphical representation of the cell stress gradient that forms across the membrane is shown.

  • Fig. 2 Governing parameters and broad applicability.

    (A) Microscopy of HEK293T cells into which FITC-labeled ssDNA was delivered through our chip. Results shown are from two independent chips. Control indicated all the same treatments for the cells except passing through the chip. Scale bar, 50 μm. BF, bright field. (B and C) Delivery efficiency (B) and cell viability (C) 16 hours after treatment were calculated for (A) as a function of fluid speed at different parameter designs; 4 or 5 μm indicates the constriction width, and 4 μm ×3 indicates cells passing through the same device three times. Error bars indicate SEM (n = 3). (D) Western blotting of PC-3 cells 48 hours after delivery with three different siRNA oligos targeting Akt1. Actin is showed as a loading control. (E) Cells from (D) were seeded in complete medium and, after 6 days, were recovered and trypsinized to count the numbers with a Countess II FL Automated Cell Counter (Life Technologies). Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (F) Delivery efficiency in different cell lines. HEK293T cells, human luminal-like MCF7 and basal-like SUM159 breast cancer cells, human SU-DHL-1 anaplastic large cell lymphoma cells, and mouse AB2.2 embryonic stem cells were delivered with plasmids encoding GFP. Untreated serves as a negative control and FuGENE HD serves as a positive control. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test.

  • Fig. 3 EGFP knockout via a microfluidic method.

    (A) Microscopy of MDA-MB-231 cells stably expressing EGFP 7 days after being delivered with plasmids encoding only Cas9 protein or both sgEGFP and Cas9 protein. Scale bar, 20 μm. (B) Percentage of cells displaying EGFP fluorescence from (A) was quantified by flow cytometry. MDA-MB-231 serves as a negative control for EGFP fluorescence signal. Error bars indicate SE (n = 3). (C) Microscopy of SU-DHL-1 lymphoma cells stably expressing EGFP 7 days after being delivered with plasmids encoding only Cas9 protein or both sgEGFP and Cas9 protein. Scale bar, 20 μm. (D) Percentage of cells displaying EGFP fluorescence from (C) was quantified by flow cytometry. SU-DHL-1 serves as a negative control for EGFP fluorescence signal. Error bars indicate SE (n = 3). (E) PCR product sequencing data for the sgEGFP-1 targeting region in SU-DHL-1 lymphoma cells. The 20–base pair (bp) target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. WT, wild type.

  • Fig. 4 Gene disruption via chip.

    Plasmids encoding both sgRNA targeting AAVS1 locus or NUAK2 and Cas9 protein were delivered into MCF7 and HeLa cells, respectively. After 7 days of cell culture, genomic DNA was extracted. PCR product sequencing for specific targeting regions was performed. (A) PCR product sequencing data for the sgAAVS1 targeting region. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. Representative sequences for indels are shown. Short black lines denote different deletions. Black arrow denotes an insertion. (B) Surveyor mutation detection assay for sgAAVS1- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S. The symbol * indicates the cleavage lane of DNA bands after cells went through the same chip three times. (C) Illustration of sgNUAK2 targeting region at the first exon. The 20-bp target sequence is shown in red; the PAM sequence is shown in blue. (D) PCR product sequencing data for the sgNUAK2 targeting region. Representative sequences for deletions are shown. Short black lines denote different deletions. (E) Surveyor mutation detection assay for sgNUAK2- and Cas9 protein–mediated indels via chip. Arrows indicate the expected positions of DNA bands cleaved by Surveyor Nuclease S.

  • Fig. 5 Microfluidic platform for cell phenotype and gene function analysis.

    (A) MCF7 cells delivered with plasmids encoding only Cas9 protein or both sgPten and Cas9 protein were cultured for 7 days and then analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. The symbol * indicates long exposure. (B) Cells (5 × 104) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days. Error bars indicate SEM (n = 3). *P < 0.005 determined by Student’s t test. (C) HeLa cells delivered with plasmids encoding only Cas9 protein or both sg53BP1 and Cas9 protein were cultured 7 days. Then, the cells were treated with 1 μM CPT for 2 hours and then examined by immunostaining with anti-53BP1 antibodies (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Survival rate of HeLa cells from (C) after control or CPT treatment was assessed by colony survival assay. Error bars indicate SEM (n = 3).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/7/e1500454/DC1

    Fig. S1. Performance of chip with different designs.

    Fig. S2. Cell stress simulation.

    Fig. S3. Flow velocity simulation.

    Fig. S4. Comparison of FuGENE HD transfection and delivery via chip.

    Fig. S5. Flow cytometric analysis of EGFP knockout cells.

    Fig. S6. Pten and 53BP1 knockout mediated by delivery via chip.

    Movie S1. Cells passing through the diamonded microconstrictions.

    Movie S2. Flow velocity simulation in the diamonded microconstriction chip.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Performance of chip with different designs.
    • Fig. S2. Cell stress simulation.
    • Fig. S3. Flow velocity simulation.
    • Fig. S4. Comparison of FuGENE HD transfection and delivery via chip.
    • Fig. S5. Flow cytometric analysis of EGFP knockout cells.
    • Fig. S6. Pten and 53BP1 knockout mediated by delivery via chip.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Cells passing through the diamonded microconstrictions.
    • Movie S2 (.avi format). Flow velocity simulation in the diamonded microconstriction chip.

    Files in this Data Supplement: