A new system that transports gene-editing tools into cells may leave other such systems in its wake. The new system, developed by American and Danish scientists, uses an ultrasound-powered nanopropeller. Instead of blades, the nanopropeller has an asymmetrically shaped gold nanowire that it churns through the fluid mosaic of the cell’s plasma membrane. Once inside the cell, the nanopropeller falls apart and frees its cargo: a gene-editing complex consisting of the CRISPR-associated protein 9 (Cas9) and a single-guide RNA (sgRNA).
Although Cas9–sgRNA complexes are widely used in genetic engineering applications, they are bulky and, hence, hard to deliver. Rather than treat these complexes like so many messages tucked inside bulbous bottles, a scientific team lead by UC San Diego’s Liangfang Zhang, Ph.D., and Joseph Wang, Ph.D., decided to try a sleek, motorized process, an acoustically driven form of active transport.
Details of their transporter’s maiden voyage appeared February 6 in the journal Angewandte Chemie, in an article entitled “Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.” This article describes how the scientists assembled a transport vehicle by attaching the Cas-9 protein/RNA complex to a gold nanowire through sulfide bridges.
These reducible linkages have an extra advantage inside tumor cells. There, the linkages are broken by glutathione, which tumor cells possess in abundance. Then, Cas9–sgRNA is released and sent to the nucleus to do its editing work, for, example, the knockout of a gene.
As a test system, the scientists monitored the suppression of fluorescence emitted by green fluorescence protein expressing melanoma B16F10 cells. Ultrasound was applied for five minutes, which accelerated the nanomotor carrying the Cas9–sgRNA complex across the membrane, accelerating it even inside the cell. Moreover, the Cas9–sgRNA complex effectively suppressed fluorescence with only tiny concentrations of the complex needed.
“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,” the article’s authors noted. “More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex.”
The authors emphasized two takeaways. First, an acoustic nanomotor that can serve as an active transport vehicle is part of relatively compact system. Second, the system is simple, requiring just a few and readily available components.
“This nanomotor-based intracellular delivery method,” the authors concluded, “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.”