Additional file 1 of Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

Supplementary Material

Recyclable Periodic Nanostructure Formed by Sublimable Liquid Crystals for Robust Cell Alignment

We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and uniaxially arranged smectic LC structures are obtained, followed by sublimation and recondensation processes, which directly produce periodic nanogrooves with dimensions of a couple of hundreds of nanometers. After treatment with osmium tetroxide (OsO4), the nanogroove can serve as a scaffold to efficiently induce directed cell growth without causing cytotoxicity, and it can be used repeatedly. Together, various cell types are applied to the nanogroove, proving the scaffold's broad applicability. Depending on the nanotopography of the LC structures, cells exhibit different morphologies and gene expression patterns, compared to cells on standard glass substrates, according to microscopic observation and qPCR. Furthermore, cell sheets can be formed, which consist of oriented cells that can be repeatedly formed and transferred to other substrates, while maintaining its organization. We believe that our cell-aligning scaffold may pave the way for the soft material field to bioengineering, which can involve fundamentals in cell behavior and function, as well as applications for regenerative medicine

An Antibody‐CRISPR/Cas Conjugate Platform for Target‐Specific Delivery and Gene Editing in Cancer

Abstract The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody‐CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2‐positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody‐conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2‐positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody‐CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2‐positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody‐mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases

Bioorthogonal CRISPR/Cas9‐Drug Conjugate: A Combinatorial Nanomedicine Platform

Abstract Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self‐delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi‐functionalized Cas9 with a drug and polymer can form self‐condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti‐cancer drug olaparib and targeting the RAD52 gene, leading to significant anti‐tumor effects in BRCA‐mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer