CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats) has emerged as a cutting-edge genome editing technique during the past decade. Viral vectors have been commonly utilized for the delivery of Cas9-sgRNA ribonucleoprotein (RNP) in-vitro. However, their ability to integrate within the host genome and limiting packing space issue limits their use for practical purposes. That is why we envision that our polymeric platform can serve as a robust non-viral vehicle for Cas9-sgRNA RNP delivery and traceless release. To achieve this goal, we have formulated protein-polymer assemblies that are formed due to covalent self-assembly of a random co-polymer, templated by Cas9 protein. Our hypothesis is that the surface exposed lysine functionalities on Cas9-sgRNA RNP can covalently react with a few polymer chains to form self-assembled structures with Cas9 at the core. A secondary crosslinker can react with remaining side-chain moieties on the polymer to form a protective sheath around the RNP, the function of which can only be revealed upon specific structural modification of the polymer because of intracellular trigger application. We have optimized several conjugation parameters to achieve ~60% encapsulation efficiency. The turn on/off feature of the assemblies has been investigated via cell-free editing assay. We have successfully shown the uptake of polymer-CRISPR conjugates inside HeLa and HEK-293 cells with final localization inside nuclei. The genome editing ability of released RNP has been shown via loss of fluorescence in GFP expressing HEK 293 cells.

Non-viral genome editing based on polymer-derived CRISPR conjugates

2019 Spring National Meeting

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