In cancer research, the Cas-9–sgRNA complex is an effective genomic editing tool, but the problem of its delivery across the cell membrane to the target (tumor) genome has not yet been satisfactorily solved. Liangfang Zhang, Joseph Wang, University of California San Diego, La Jolla, CA, USA, and colleagues have developed an active nanomotor for the efficient transport, delivery, and release of this gene scissoring system. Their nanovehicle is propelled towards its target by ultrasound.
Genomic engineering as a promising cancer therapeutic approach has experienced a surge since the discovery of the adaptive bacterial immune defense system CRISPR and its potential as a gene editing tool over a decade ago. Engineered CRISPR systems for gene editing now contain two main components, a single guide RNA or sgRNA and a Cas-9 nuclease. While the sgRNA guides the nuclease to the specified gene sequence, Cas-9 nuclease performs its editing with surgical efficiency. However, the delivery of the large machinery to the target genome is still problematic.
Ultrasound-Propelled Gold Nanowires
The team has used ultrasound-propelled gold nanowires as an active transport/release vehicle for the Cas9–sgRNA complex over the membrane. Gold nanowires may cross a membrane passively, but thanks to their rod- or wirelike asymmetric shape, active motion can be triggered by ultrasound.
“The asymmetric shape of the gold nanowire motor, given by the fabrication process, is essential for the acoustic propulsion,” the researchers state. They assembled the vehicle by attaching the Cas-9 protein/RNA complex to the gold nanowire through sulfide bridges. These reduceable linkages have the advantage that inside the tumor cell, the bonds are broken by glutathione, a natural reducing compound enriched in tumor cells. The 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 team observed their Cas9–sgRNA complex effectively suppressing fluorescence with only tiny concentrations of the complex needed.
Thus, both the effective use of an acoustic nanomotor as an active transporter and the small payload needed for efficient gene knockout are intriguing results of the study. The simplicity of the system, which uses only a few readily available components, is also remarkable.
- Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors,
Malthe Hansen-Bruhn, Berta Esteban-Fernández de Ávila, Mara Beltrán-Gastélum, Jing Zhao, Doris E. Ramírez-Herrera, Pavimol Angsantikul, Kurt Vesterager Gothelf, Liangfang Zhang, Joseph Wang,
Angew. Chem. Int. Ed. 2018.