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

CRISPR/Cas9系统已成为最令人兴奋的基因组编辑工具，广泛应用于基础研究、农业和生物医学工程。然而，将CRISPR/Cas9组分有效地递送到靶细胞中仍然是一个挑战。大多数CRISPR/Cas9传递系统依赖于物理或病毒方法。物理方法存在细胞活力低和细胞依赖效率低的问题。病毒载体受到包装能力、基因突变和免疫原性的限制。相比之下，使用纳米颗粒（NPs）的新兴非病毒递送方法具有克服这些限制的潜力。然而，许多挑战依然存在。1)效率：非病毒传递系统的效率仍然低于病毒传递系统。影响输送效率的关键特性之一是NPs的稳定性。目前用于CRISPR/Cas9递送的非病毒NP系统，包括最常用的Lipofectamine，主要是通过弱物理相互作用形成的。这些NPs稳定性差，容易导致CRISPR/Cas9过早释放，影响传递效率和基因组编辑效率。NPs的稳定性是由其内在的物理化学性质决定的。优化这些特性以实现NPs的卓越稳定性对于CRISPR/Cas9传递至关重要。2)精确性：CRISPR/Cas9广泛应用的另一个障碍是缺乏能够靶向特定细胞/组织并编程基因组编辑的传递系统。它需要智能的非病毒NPs，能够优先在目标位点传递和激活CRISPR/Cas9组分。NPs的表面物理化学性质，如大小和化学成分，极大地影响细胞/组织靶向性。此外，与期望的刺激反应化学相结合的传递系统可以支持“按需”基因组编辑。因此，我的研究项目的长期目标是设计下一代传递系统，以实现高效和精确的基因组编辑。我在未来五年的短期目标是：目标1：设计和优化具有调谐物理化学性质的共价交联聚合物纳米凝胶（CCPNs），用于普遍递送CRISPR/Cas9 DNA， mRNA和RNP。目的2：研究CRISPR/Cas9 CCPNs表面物理化学性质对细胞/组织选择性靶向的影响。目标3：为可控的CRISPR/Cas9基因组编辑设计刺激反应性ccpn。本研究将为精准高效的CRISPR/Cas9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生物学研究、农业和疾病治疗等不同领域的应用。化学选择和材料设计对纳米生物相互作用的影响的新发现和理解将进一步影响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 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.