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

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.


Supplementary Note 1. Estimation of pulse / echo and spatial resolution of a high
frequency ultrasonic transducer. Pulse / echo time and frequency responses were measured to find a center frequency (f) of a high frequency ultrasonic transducer using a custom-made ultrasound biomicroscopy (UBM) system. The schematic diagram of the UBM is shown in Supplementary Fig. 1. The UBM system was composed of a main computer, a pulser / receiver (DPR500 pulser / receiver, Imaginant Inc. Pittsford, New York), a 3D axis stage (ILS100HA, Newport, Irvine, California) with a stage controller (ESP301-3N, Newport, Irvine, California), and an analog-to-digital acquisition card (A/D card, CS122G1, DynamicsSignals LLC., Lockport, Illinois). A quartz block was used as a reflecting target for the pulse / echo measurements at the focus of the high frequency ultrasonic transducer. The high frequency ultrasonic transducer was mounted on a 3D axis stage. Pulses from a pulser / receiver excited the high frequency ultrasonic transducer with 200 Hz pulse repetition frequency (PRF). The reflected echoes were received by the same ultrasonic transducer and amplified by the same pulser / receiver ( Supplementary Fig.   1). The pulses and amplified echoes were saved and post-processed by a custom-built Matlab where cut-off1 and cut-off2 are the -6 dB frequencies from the estimated spectra as shown in For the axial and lateral resolutions measurements, the high frequency ultrasonic transducer was translated with a step size of 1 μm to acquire the scan lines required to obtain 2D cross-sectional images of a tungsten wire target, as shown in Supplementary Fig. 1 which determines the fnumber (Fig. 1A). A large aperture and fnumber will occupy more spaces.
Therefore, if acoustic-transfection and microfluidic channels are combined for intracellular delivery, a small aperture and fnumber are desired. Once the fabrication of the ultrasonic transducers is completed, the strength of acoustic-transfection is easily controlled by changing the input parameters (Vp, tw, PRT, and NP) of the electrical signal using an electronic control system such as a power amplifier and function generator (Fig. 1C2).
The level of the normalized pressure ( ̅ , nondimensionalized) at the focus of an ultrasonic transducer is simulated in Supplementary Fig. 2. The angular spectrum method was used for simulation 1 . Normalized pressure is where is the real pressure at the focus, 0 is the density of water, 0 is the speed of sound, and is the velocity of the aperture. An ultrasonic transducer with a larger ap, smaller fnumber, and higher frequency generates a higher level of pressure at the focus, as shown in Supplementary where fnumber is the ratio of focal distance to the aperture as shown in Fig. 1A, is the wavelength of the acoustic sound waves, is the speed of sound in water (1.5 mm/μs), f is the center frequency of the acoustic sound waves (Equation S1 in Supplementary Note 1), S is the area of the aperture, defined as ( ) 2 , and d is the focal distance (Fig. 1A). In this study, we designed ap, d, and f of the ultrasonic transducer to be 1 mm, 1 mm, and 150 MHz, respectively, resulting in values for and to be 10 μm and 79, respectively.  Playback is at 7 frames per second. EGFP intensities were not modified.

Supplementary
Supplementary Video 2. The mNeonGreen protein delivery into a single-cell using acoustictransfection. Time-lapse imaging of a mNeonGreen acoustic-transfected HeLa cell. Images were taken 1 hour after acoustic-transfection every 20 minutes for 7 hours. Playback is at 7 frames per second. The mNeonGreen intensities were not modified.
Supplementary Video 3. Calcein efflux due to diffusion after an acoustic pulse on a target cell.
Time-lapse imaging of calein loaded HeLa cells on the left and a plot of calcein fluorescence intensity of a target cell on the right. After calcein was loaded on the cells, an acoustic pulse was applied to only one HeLa cell. Shortly after an acoustic pulse was applied, calcein intensity decreased, indicating the efflux of calcein due to diffusion through transient holes on the cell membrane. Images were taken every 0.5 seconds. Playback is at 3.5 frames per second. No intensity modification was applied.

Reference
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