Supramolecular Nanosubstrate‐Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion. Issue 28 (8th June 2021) - Ban, Qian
Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)‐associated protein 9 (CRISPR/Cas9) is an efficient and precise gene‐editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate‐mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular‐uptake of the RNP‐laden nanoparticles are examined. Dose‐ and time‐dependent CRISPR/Cas9‐mediated gene disruption is further examined in a green fluorescent protein (GFP)‐expressing U87 cell line (GFP‐U87). The utility of an optimized SMNP formulation in co‐delivering Cas9 protein and two sgRNAs that target deletion of exons 45–55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells. Abstract : Supramolecular nanosubstrate‐mediated delivery facilitates intracellular entry of Cas9 ribonucleoprotein into target cells. Local enrichment of supramolecular nanoparticles from the surrounding medium onto nanowires via molecular recognition enables highly efficient clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR/Cas9) gene disruption and deletion. This platform offers a general clinical therapeutic solution for genetic diseases, such as Duchenne muscular dystrophy.