Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

Abstract

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.