Scaffold-based delivery of CRISPR/Cas9 ribonucleoproteins for genome editing

• Main Authors: Chooi, Wai Hon, Chin, Jiah Shin, Chew, Sing Yian
• Other Authors: School of Chemical and Biomedical Engineering
• Format: Article
• Language: English
• Published: 2021
• Subjects: Engineering::Chemical engineering; Gene delivery; Electrospinning
• Online Access: https://hdl.handle.net/10356/145891
• Institution: Nanyang Technological University

The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.