A programmable, cell-agnostic DNA nano-technology platform for CRISPR gene editing

CRISPR-Cas9基因编辑使其开发者获得了2020年诺贝尔化学奖，因为这项技术正在对生物、生物技术和医学研究产生巨大影响。特别是，CRISPR基因编辑是治疗癌症和其他疾病的下一代基于细胞的疗法的核心，在这种疗法中，患者的一些细胞被提取，经过基因改造以实现特定功能，然后重新注入患者体内。然而，这一过程既耗时又昂贵，限制了这些潜在的挽救生命的疗法的传播。成本高昂背后的原因之一是，人们可以向细胞输送执行CRISPR基因编辑所需的生物机械的低效率，无论是在规模上还是在没有过度毒性(导致细胞死亡)的情况下。在这个项目中，我们将开发一种新的替代方法，将CRISPR机械输送到体外的哺乳动物细胞。我们的方法将依赖于专门设计的载体，我们称之为编辑体。这些是由类脂膜构建的微观包膜，类似于细胞膜，含有大量的CRISPR机器。要使编辑体将机器运送到靶细胞，两者必须融合。我们将通过用人造的“融合基因”纳米机器装饰编辑体和靶细胞来诱导融合，通过相互结合使细胞和编辑体的膜在非常短的距离内，最终使它们合并。融合纳米结构将由合成的DNA分子构建，鉴于碱基配对相互作用的高度选择性和可编程性，这特别适合于工程纳米设备。我们预计编辑体技术将对我们在体内进行高通量、高效的CRISPR基因编辑的能力产生直接和深远的影响，从而对依赖它的治疗技术的可获得性和经济可持续性产生直接和深远的影响。此外，我们还将阐明脂质膜稳定性、融合和DNA纳米结构调节它们的(生物)物理的基本方面。

CRISPR-Cas9 gene editing has warranted its developers the 2020 Nobel Prize for Chemistry, in view of the massive impact that this technology is having on biological, biotechnological and medical research. In particular CRISPR gene editing is central to next generation cell-based therapies to treat cancer and other diseases, in which some of the patient's cells are extracted, genetically modified to fulfil specific functions, and then re-injected into the patient. This process is however time consuming and very costly, limiting the diffusion of these potentially life-saving therapies. One of the reasons behind the prohibitive costs is the low efficiency with which one can deliver to the cells the biological machinery required to perform CRISPR gene editing, both at scale and without excessive toxicity (leading to cell death).In this project, we will develop a novel and alternative approach to delivering CRISPR machinery to mammalian cells in vitro. Our approach will rely on specifically designed vectors, which we dub Editosomes. These are microscopic enclosures constructed from lipid membranes, similar to cell membranes, and containing large quantities of the CRISPR machinery.For Editosomes to deliver the machinery to the target cells the two would have to fuse. We will induce fusion by decorating both Editosomes and the target cells with artificial "fusogenic" nanomachines, that by binding to each other bring the cell and Editosome membranes to within a very short distance, ultimately making them merge. The fusogenic nanostructures will be constructed from synthetic DNA molecules, which are particularly suitable for engineering nanodevices in view of the very high selectivity and programmability of the base-pairing interactions.We envisage that Editosome technology will have a direct and profound impact on our ability to perform high-throughput, efficient, CRISPR gene editing in vivo, and thus on the accessibility and economic sustainability of the therapeutic technologies that rely on it.Additionally, we will clarify fundamental aspects of the (bio)physics underling lipid membrane stability, fusion, and the ability of DNA nanostructures to modulate them.