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

用于 CRISPR 基因编辑的可编程、与细胞无关的 DNA 纳米技术平台

• 批准号: EP/V048058/1
• 负责人: Lorenzo Di Michele
• 金额: $ 25.69万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FV048058%2F1
• 关键词: programmable; cell; agnostic; DNA; nano

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.

鉴于 CRISPR-Cas9 基因编辑技术对生物、生物技术和医学研究产生的巨大影响，其开发人员获得了 2020 年诺贝尔化学奖。特别是，CRISPR基因编辑是治疗癌症和其他疾病的下一代细胞疗法的核心，其中提取患者的一些细胞，进行基因改造以实现特定功能，然后重新注射到患者体内。然而，这个过程非常耗时且成本高昂，限制了这些可能挽救生命的疗法的传播。成本高昂的原因之一是，向细胞传递进行 CRISPR 基因编辑所需的生物机器的效率很低，既要大规模，又不会产生过多的毒性（导致细胞死亡）。在这个项目中，我们将开发一种新颖的替代方法，在体外将 CRISPR 机器传递给哺乳动物细胞。我们的方法将依赖于专门设计的载体，我们将其称为编辑体。这些是由脂质膜构建的微观外壳，类似于细胞膜，并且包含大量的 CRISPR 机器。为了将机器传递到目标细胞，编辑体必须将两者融合。我们将通过用人工“融合”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膜达到非常短的距离，最终使它们融合。融合纳米结构将由合成 DNA 分子构建，鉴于碱基配对相互作用的极高选择性和可编程性，特别适合工程纳米器件。我们预计，Editosome 技术将对我们的能力产生直接而深远的影响。在体内进行高通量、高效的 CRISPR 基因编辑，从而提高依赖它的治疗技术的可及性和经济可持续性。此外，我们将阐明脂膜下的（生物）物理学的基本方面DNA 纳米结构的稳定性、融合性以及调节它们的能力。

项目成果

Modulating membrane fusion through the design of fusogenic DNA circuits and bilayer composition.

通过融合 DNA 电路和双层组成的设计来调节膜融合。

• DOI: http://dx.10.1039/d2sm00863g
• 发表时间: 2022
• 期刊: Soft matter
• 影响因子: 3.4
• 作者: Paez

Sculpting DNA-based synthetic cells through phase separation and phase-targeted activity

通过相分离和相靶向活性来塑造基于 DNA 的合成细胞

• DOI: 10.1101/2023.03.17.533162
• 发表时间: 2023-03-21
• 期刊: bioRxiv
• 作者: Layla Malouf;Diana A. Tanase;Giacomo Fabrini;Miguel Paez;Adrian Leathers;M. Booth;L. Di Michele
• 通讯作者: L. Di Michele