Core D GET iN

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Genetic modification, such as gene knockdown, overexpression or editing, is the most effective way to elucidate gene function, which has the potential to advance therapeutic intervention. The delivery of genetic material into primary skin cells, however, is notoriously difficult and delivery through the epidermal barrier is a great challenge. The Gene Editing, Transduction, and Nanotechnology (GET iN) Core will apply to cutaneous research the most advanced and innovative approaches for genetic manipulation. These include CRISPR/Cas9 technology and both viral and non-viral methods for the delivery into skin of DNA, RNA, and other biomolecules. Specifically, the Core will generate high titer viral stocks for gene overexpression and silencing for in vitro and in vivo applications. The Core will offer viruses for constitutive/inducible expression under ubiquitous or skin cell-specific promoters and viral expression vectors for a broad range of specific applications. These include: i) transcription factor luciferase reporters; ii) cell labeling/tracking and live cell imaging; iii) immortalization of primary skin cells; iv) generation of induced pluripotent stem (iPS) cells; and v) iPS cell reprogramming. The Core will provide reagents for DNA editing and epigenetic modifications using CRISPR-based technologies in primary skin cells and cell lines, using both viral delivery of Cas9 and gRNAs (under constitutive or inducible promoters), and non- viral delivery of recombinant Cas9 protein + synthetic gRNAS in the form of RNA – protein complexes (RNPC). We will also offer CRISPR applications (e.g., CRISPRa and CRISPRi) using nuclease-deactivated Cas9 (dCas9) to regulate gene promoter activity. We will develop novel non-viral delivery methods for gene overexpression/silencing in primary skin cells, skin-derived cell lines and skin in vivo. The Core has adapted the novel Nanofountain Probe Electroporation (NFPE), a micro-fluidic device developed at Northwestern for targeted delivery to single cells, for introducing viruses, DNA, RNA, proteins, dyes, and drugs. The Core will also facilitate the use of spherically oriented nanocarriers (e.g., spherical nucleic acids and high density lipoprotein nanoparticles) for intracellular and transcutaneous DNA, RNA and drug delivery to cells and skin, respectively. SBDRC members will also be able to deliver DNA and RNA to skin cells using the Gene Gun. We will establish archives and biorepositories, and offer consultation on delivery options into skin cells and skin. Having a wide range of customized viral vectors and unique reagents, novel CRISPR/Cas9 services, and innovative nanotechnology/NFPE delivery approaches, this Core will provide the most advanced tactics for genetic manipulation in 2D and 3D skin cultures, as well as skin in vivo. Our services, and collaboration with other SBDRC and Northwestern Cores, will further enhance skin biology and diseases research at NU, and ultimately facilitate the bench to bedside translation of research.