Engineering Controllable Non-Viral Systems for Efficient and Precise CRISPR/Cas9 Delivery

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The CRISPR/Cas9 system has emerged as the most exciting genome editing tool for broad applications in fundamental research, agriculture, and biomedical engineering. However, effective delivery of CRISPR/Cas9 components into target cells remains a challenge. Most CRISPR/Cas9 delivery systems rely on physical or viral approaches. Physical methods suffer from low cell viability and cell-dependent efficiency. Viral vectors are limited by restricted packaging capacities, genetic mutations and immunogenicity. In comparison, the emerging non-viral delivery methods using nanoparticles (NPs) have the potential to overcome these limitations. However, many challenges remain. 1) Efficiency: The efficiency of non-viral delivery systems remains lower than the viral ones. One of the key properties that can affect delivery efficiency is the stability of NPs. Current non-viral NP systems for CRISPR/Cas9 delivery, including the most commonly used agent Lipofectamine, are primarily formed through weak physical interactions. Poor stability of these NPs can easily lead to premature release of CRISPR/Cas9, compromising delivery efficiency and genome editing efficiency. Stability of NPs is determined by their intrinsic physicochemical properties. Optimizing these properties to enable superior stability of NPs is essential for CRISPR/Cas9 delivery. 2) Precision: Another obstacle to broad application of CRISPR/Cas9 is the lack of delivery systems that can target specific cells/tissues and program genome editing. It requires intelligent non-viral NPs that can preferentially deliver and activate CRISPR/Cas9 components at targeted sites. Surface physicochemical properties of NPs, such as size and chemical compositions, greatly impact cell/tissue targeting. Additionally, delivery systems integrated with desired stimuli-responsive chemistry can favor "on-demand" genome editing. Hence, the long-term objective of my research program is to engineer next-generation delivery systems to achieve efficient and precise genome editing. My short-term aims in the next five years are: Aim 1: Design and optimize covalently crosslinked polymeric nanogels (CCPNs) with tuned physicochemical properties for universal delivery of CRISPR/Cas9 DNA, mRNA, and RNP. Aim 2: Investigate effects of surface physicochemical properties of CRISPR/Cas9 CCPNs on selective cell/tissue targeting. Aim 3: Engineer stimuli-responsive CCPNs for controlled CRISPR/Cas9 genome editing. This research will establish a new generation of non-viral delivery systems for precise and efficient CRISPR/Cas9 genome editing. The resultant technology will significantly broaden the application of genome editing tools in diverse settings across fundamental biological research, agriculture, and disease therapeutics. New discoveries and understanding in the effects of chemical selection and materials design on nano-bio interactions will further impact development of advanced drug delivery systems and biomaterials.