Engineering Next-Generation Nanoformulations for Bioresponsive CRISPR/Cas9 Delivery

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The CRISPR/Cas9 system has emerged as the most exciting genome editing tool for widespread applications in fundamental biological research, bioengineering, agriculture, and healthcare. However, the greatest challenge to its deployment for genetic engineering is the lack of safe, effective, and selective delivery of CRISPR/Cas9 elements.Most CRISPR/Cas9 delivery systems still rely on viral approaches. Although viral vectors offer good delivery performance, they are limited by restricted packaging capacities and safety concerns. Non-viral delivery via nanoparticles (NPs) has the potential to overcome these limitations. While recent efforts have been focused on designing desired CRISPR/Cas9 non-viral vectors, there are still challenges lying ahead. 1) Efficiency: The efficiency of the existing non-viral NPs for CRISPR/Cas9 remains modest. These NPs are formed mainly by self-assembly via weak interactions with poor stability, which leads to premature release of CRISPR/Cas9, thus compromising delivery efficiency. 2) Precision: Current non-viral delivery approaches also lack the ability to target specific cells/tissues for precise genome editing. Therefore, it is extremely valuable to develop a non-viral delivery system that can simultaneously achieve high efficiency and precision in CRISPR/Cas9-mediated genome editing.Calcium Carbonate (CaCO3) NPs have received wide attention in drug delivery, due to their excellent biocompatibility, simple fabrication process, and most importantly, superior stability. Advances in nanotechnology have led to the development of polymer/CaCO3 (pCaCO3) hybrid NPs with controllable physicochemical properties. These advantages place pCaCO3 NPs as an ideal candidate as a drug nanocarrier for CRISPR/Cas9, offering solutions to achieve efficient and precise genome editing, which, however, has not been previously investigated. Hence, the objective of this proposal is to engineer pCaCO3 NP systems for bioresponsive CRISPR/Cas9 delivery to achieve efficient and precise genome editing. We have three specific aims.Aim 1: Design and optimize pCaCO3 NPs for CRISPR/Cas9 ribonucleoprotein (RNP) delivery. Aim 2: Investigate effects of surface physicochemical properties of RNP@pCaCO3 NPs on selective cell/tissue targeting. Aim 3: Engineer pCaCO3 NPs for co-delivering RNP and donor DNA template for gene correction. This research will establish a new generation of non-viral delivery systems for precise and efficient genome editing. The resultant technology will significantly broaden the application of genome editing tools in diverse settings across fundamental science to applied research. New discoveries in the effects of chemical selection and materials design on nano-bio interactions will further contribute to the development of advanced drug delivery systems and biomaterials.