Abstract
Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.
Highlights
Intracellular delivery of macromolecules into target cells is an essential and fundamental procedure to modulate cell functions for research and clinical applications
We demonstrate the intracellular delivery of DNA plasmids, messenger RNA (mRNA), recombinant proteins, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] systems using acoustic-transfection to visualize and genetically edit cells by homologous-directed repair (HDR)
The results show potential advantages over other transfection techniques when studying cell-to-cell interaction by combining acoustic-transfection with fluorescence resonance energy transfer (FRET)-based biosensors (BS) and live cell imaging techniques to visualize important molecular events and cell reprogramming by repeatedly delivering multiple recombinant proteins or CRISPR-Cas[95, 6]
Summary
Intracellular delivery of macromolecules into target cells is an essential and fundamental procedure to modulate cell functions for research and clinical applications. We demonstrated a potential for intracellular delivery of small exogenous molecules such as propidium iodide (MW: 0.7 kDa) and 3 kDa dextran using acoustic-transfection[20] These delivered compounds are relatively inert and do not allow the manipulation of target cells. We demonstrate the intracellular delivery of DNA plasmids, mRNAs, recombinant proteins, and CRISPR-Cas[9] systems using acoustic-transfection to visualize and genetically edit cells by homologous-directed repair (HDR). This demonstration proves that acoustic-transfection can deliver a wide range of biologically active macromolecules noninvasively and remotely by utilizing high frequency ultrasound. The results show potential advantages over other transfection techniques when studying cell-to-cell interaction by combining acoustic-transfection with FRET-based biosensors (BS) and live cell imaging techniques to visualize important molecular events and cell reprogramming by repeatedly delivering multiple recombinant proteins or CRISPR-Cas[95, 6]
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