53 / 2019-07-24 17:55:39
Wearable flexible nano-transfection device for on-skin gene editing with CRISPR-Cas9
nano-transfection,CRISPR-Cas9,Wearable device
摘要录用
Zaizai Dong / Beihang University
Yongcun Hao / Northwestern Polytechnical University
Chandani Chitrakar / University of North Texas
Honglong Chang / Northwestern Polytechnical University
Lingqian Chang / Beihang University
Wearable flexible nano-transfection device for on-skin gene editing with CRISPR-Cas9

Z. Dong1,2, Y. Hao3, C. Chitrakar4, H. Chang3, L. Chang1,2,4*

1 School of Biological Science and Medical Engineering, Beihang University, Beijing, 100083, China
2 Institute of Nanotechnology for Single Cell Analysis (INSCA), Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, 100083, China
3 Department of Microsystems, Northwestern Polytechnical University, Xian, China, 710065
4 Department of Biomedical Engineering, University of North Texas, Denton, TX, 76207, USA

Contact email address: changlingqian1986@buaa.edu.cn
Introduction
Recent advances in CRISPR-Cas9 techniques have shown possibilities for the “in-body”, patient-specific in vivo gene editing, transcriptional modulation and live-cell imaging1. However, currently available gene delivery techniques face two major hurdles for direct on-skin transfection of CRISPR-Cas9. First, the transfection efficiency is extremely low (< 10 %) for large molecular weights (> 6 kbps, e.g. RNA-guided CRISPR-associated Cas9 protein). Furthermore, targeting to local cells is challenging, impossible for dynamic tracking and analysis. Both issues eventually lead to its difficulty in clinical trials2.
Materials and methods
The device was fabricated on polyimide substrate, which could be easily adjusted for the curves of different skin (Fig. 1a). The wearable device integrated multiple functions on one chip. The core of gene delivery is achieved upon nano-electroporation, which consists of array of micro-electrode (width: 20 μm) in connection with nano-wells (diameter: 600 nm) where the CRISPR-Cas9 is placed for delivery. A micro-needle inserted into the skin is treated as the top electrode. Patching on the skin, the electric field is applied between the top electrode and bottom micro-electrode for cell electroporation (Fig. 1b). The nano-well feature could accurately tune the electric field on the cell membrane, which greatly reduces the area affected from the electroporation while improving the cell safety. Uniquely, the concentrated electric field via nano-well “electrophoretically” drives the charged-cargo (i.e. CRISPR and Cas9 proteins) into cells with high speed (Fig. 1c)3, which achieves precise dose control and high transfection efficiency. The power for cell electroporation is supplied by a wireless communication zone constituted with an ultra-thin magnetic spiral antenna and a near-field communication (NFC) chip. The spiral antenna is made by gold (Au) (thickness: 20 nm) deposition in photolithographic patterning (Fig. 1d). NFC technology, remotely controlled by a cell phone, is applied to wirelessly control the cell electroporation.
Results
Aiming to melanoma therapy, we delivered plasmids CRISPR-Cas9 cargo into A375 cells on 3D EP platform as preliminary case study. The reporter gene expression confirmed that our platform achieved high delivery efficiency (>90%) for macromolecular plasmids (> 9 kb), significantly higher than BEP (~ 20%) (Fig 2).

Fig. 1 A flexible intracellular delivery nano-device patched on the skin for precise gene delivery into epidermal cells. (a) The nano-features were patterned on polyimide substrate, which could be easily adjusted to various skins. (b) The schematic illustrates the nano-well array connect the epidermal cells. The electric field is applied between bottom micro-electrode array (bottom electrode) and a top electrode (micro-needle) for electroporating cells. (c) numerical simulation shows the nano-well can concentrate the electric field within a nanoscale-region on the cell membrane, providing a safe potential drop (less than 5 V) for electroporation. (d) The electroporation-assisted gene delivery is powered by wireless communication zone (i.e. a spiral antenna and a NFC chip).


Fig. 2 (a) DAPI and (b) GFP reporter gene fluorescence shows the high-throughput transfection results. Scale bar: 200 µm. (c) The efficiency of nano-chip is significantly higher than commercial electroporation. P-value < 0.01; N =3.

Conclusions
In this work, we report a novel flexible intracellular delivery nano-device which can be patched on the skin for precise gene delivery into epidermal cells. This simple implement to flexible gene transfection nano-device, based on cleanroom nano-fabrication techniques, provide a capability of direct, deterministic in vivo gene editing into epidermal cells on the skin.

References
1. Knight SC, Xie L, Deng W et al. (2015) Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science, 350: 823-826.

2. Gallego-Perez D, Pal D, Ghatak S et al. (2017) Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nat. Nanotechnol. 12: 974-979.

3. Chang L, Bertani P, Gallego-Perez D et al. (2016) 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control. Nanoscale, 8: 243-252.
重要日期
  • 会议日期

    09月05日

    2019

    09月06日

    2019

  • 06月05日 2019

    初稿截稿日期

  • 09月06日 2019

    注册截止日期

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