Engineering Next-Generation Nanoformulations for Bioresponsive CRISPR/Cas9 Delivery

CRISPR/Cas9系统已经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。然而，将其应用于基因工程的最大挑战是缺乏安全、有效和选择性地递送CRISPR/Cas9元件。大多数CRISPR/Cas9递送系统仍然依赖病毒方法。尽管病毒载体提供了良好的传递性能，但它们受到包装容量和安全问题的限制。通过纳米颗粒(NPs)的非病毒传递有可能克服这些限制。虽然最近的努力集中在设计所需的CRISPR/Cas9非病毒载体上，但前方仍有挑战。1)效率：CRISPR/CAS9现有的非病毒NPs的效率仍然不高。这些纳米粒子主要是通过弱相互作用自组装形成的，稳定性较差，导致CRISPR/Cas9过早释放，从而影响递送效率。2)精确度：目前的非病毒传递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，开发一种非病毒递送系统在CRISPR/Cas9介导的基因组编辑中同时实现高效和精确是非常有价值的。碳酸钙(CaCO3)纳米粒子因其良好的生物相容性、简单的制备工艺以及最重要的是优异的稳定性而在药物递送方面受到广泛的关注。纳米技术的进步导致了聚合物/碳酸钙(PCaCO3)杂化纳米粒子的发展，这些纳米粒子具有可控的物理化学性质。这些优势使pCaCO3纳米粒子成为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，这一点以前还没有得到研究。因此，这项提议的目标是设计用于生物响应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。目标1：设计和优化用于CRISPR/Cas9核糖核蛋白(RNP)传递的pCaCO3纳米粒。目的：研究RNP@pCaCO3纳米粒子表面物理化学性质对细胞/组织选择性靶向的影响。目的3：设计pCaCO3纳米粒，用于共传递RNP和供体DNA模板以进行基因校正。这项研究将建立新一代非病毒传递系统，用于精确和高效的基因组编辑。由此产生的技术将极大地扩大基因组编辑工具在不同环境中的应用，从基础科学到应用研究。在化学选择和材料设计对纳米生物相互作用的影响方面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.