Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound

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.

Development and Characterization of Ultrasound Activated Lipopolyplexes for Enhanced Transfection by Low Frequency Ultrasound in In Vitro Tumor Model

“…The cell uptake of lipopolyplexes is based on its architecture, average particle size, and zeta potential of the complex formed. [ 38 ] Unlikely, the transfection of DPPC/CH/DPPG/PEG40S lipopolyplexes (LPP2) at mass ratio 0.5 was increased up to 20‐fold than polyplexes. The lower transfection efficiency was observed because liposomes may not coat polyplexes completely at mass ratio < 0.5, while a decrease in transfection efficiency at mass ratio > 0.5 could be due to aggregation of the particles at lower zeta potential.…”

“…[ 40,41 ] Taken together, the ultrasound enhances the transfection of ultrasound activated lipopolyplexes. [ 38,42 ] There is enhanced cell transfection of the carrier when the time difference between transfection and ultrasound treatment is ≥1 h.…”

Acoustic mechanogenetics

“…We have developed an ultrasound technique to deliver macromolecules into target cells in vitro [43, 44]. Ultra-high frequency ultrasound beam can mechanically disrupt cell membrane to increase permeability of cells.…”

FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound

“…Single cell targeting and manipulation can be realized by HFU due to its focusing ability. When the frequency of HFU exceeds 150 MHz, the size of the focal area approaches that of a subcellular region smaller than 10 μm [ 26 , 27 ]. HFU can be used to mimic the physiological cues that trigger changes in gene expression and cell behavior.…”