Engineering Next-Generation Nanoformulations for Bioresponsive CRISPR/Cas9 Delivery

CRISPR/Cas9系统已成为最令人兴奋的基因组编辑工具，广泛应用于基础生物学研究、生物工程、农业和医疗保健领域。然而，将其应用于基因工程的最大挑战是缺乏安全、有效和选择性地传递CRISPR/Cas9元件。大多数CRISPR/Cas9传递系统仍然依赖于病毒方法。虽然病毒载体提供了良好的传递性能，但它们受到有限的包装能力和安全问题的限制。通过纳米颗粒（NPs）的非病毒递送具有克服这些限制的潜力。虽然最近的努力集中在设计理想的CRISPR/Cas9非病毒载体上，但仍然存在挑战。1)效率：现有的CRISPR/Cas9非病毒NPs的效率仍然不高。这些NPs主要通过弱相互作用自组装形成，稳定性差，导致CRISPR/Cas9过早释放，从而影响递送效率。2)精确性：目前的非病毒递送方法也缺乏针对特定细胞/组织进行精确基因组编辑的能力。因此，在CRISPR/ cas9介导的基因组编辑中，开发一种能够同时实现高效率和高精度的非病毒传递系统具有极其重要的价值。碳酸钙（CaCO3） NPs具有良好的生物相容性、制备工艺简单、稳定性好等优点，在给药领域受到广泛关注。纳米技术的进步导致了具有可控物理化学性质的聚合物/CaCO3 （pCaCO3）杂化NPs的发展。这些优势使pCaCO3 NPs成为CRISPR/Cas9药物纳米载体的理想候选者，为实现高效和精确的基因组编辑提供了解决方案，然而，这在以前还没有被研究过。因此，本提案的目标是设计用于生物反应性CRISPR/Cas9递送的pCaCO3 NP系统，以实现高效和精确的基因组编辑。我们有三个具体目标。目的1：设计和优化用于CRISPR/Cas9核糖核蛋白（RNP）递送的pCaCO3 NPs。目的2：研究RNP@pCaCO3 NPs的表面物理化学性质对选择性细胞/组织靶向的影响。目标3：设计pCaCO3 NPs，用于共同递送RNP和供体DNA模板，用于基因校正。这项研究将为精确和高效的基因组编辑建立新一代非病毒传递系统。由此产生的技术将显著拓宽基因组编辑工具在基础科学和应用研究等不同领域的应用。化学选择和材料设计对纳米生物相互作用影响的新发现将进一步促进先进药物输送系统和生物材料的发展。

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.