Abstract
Focused ultrasound (FUS) in combination with microbubbles (MBs) can induce cavitation-mediated plasma membrane permeabilization in nearby cells, thus permitting entry to otherwise impermeable macromolecules. Cas9 is an endonuclease protein currently at the forefront of gene editing due to its efficiency, ease of use, and low cost. In complex with single guide RNA (sgRNA), Cas9 can target specific gene sequences to cause double-stranded breaks, interrupting gene function in the process. Cas9 is best delivered as a ribonucleoprotein (RNP) for the most effective results, however, suffers from inefficient delivery methods for in-vivo applications due to its large size (160 kDa). FUS and microbubbles can be an effective alternative to currently applied systems (e.g., adeno-associated vectors) to deliver Cas9:sgRNA RNPs for CRISPR-mediated knockout. Currently, we are exploring the use of FUS for Cas9-mediated knockout of EGFP in both EGFP-expressing human induced pluripotent stem cells (hiPSC) and human cardiomyocytes. Treatment of hiPSC under acoustic conditions (1 MHz, 1000 cycles, 5 ms intervals, >208 kPa) suitable for cavitation-mediated sonoporation led to EGFP knockout in hiPSCs. By modulating FUS parameters and setup, we can optimize delivery of Cas9 as an RNP for treatment of genetic diseases, such as hypertrophic cardiomyopathy.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
