Enhanced CRISPR gene editing in pluripotent stem cells using carbon nanotube arrays

摘要 所提出的研究的目标是提高聚类规则间隔短回文的效率 在人类多能干细胞（PSC），包括胚胎干细胞中进行基于CRISPR的基因编辑 在一些实施方案中，所述干细胞包括胚胎干细胞（ESC）和诱导多能干细胞（iPSC）。目前，CRISPR/Cas9在靶向治疗方面显示出巨大的潜力。 基因编辑，但在PSC中仍然具有挑战性，因为它们对常规转染非常难治 与其他原代细胞类型和细胞系相比。这对目标群体构成了重大障碍。 PSC的遗传操作。所需要的是一种快速、有效、需要较少输入的转染方法， 细胞，并且可以以最小的毒性将DNA/RNA/蛋白质复合物引入PSC中。 AGTC开发了一种新的方法，使用设备将生物分子有效地引入哺乳动物细胞 由紧密堆积和排列的碳纳米管（CNT）阵列组成，以实现高效转移 具有低细胞毒性。AGTC还为这些CNT器件开发了一种可扩展的纳米制造工艺 使用基于模板的化学气相沉积（CVD）来生产由数千个200 nm- 直径中空碳纳米管（CNT）嵌入在13 mm直径的基础上，可与标准 组织培养板。 在这项提案中，AGTC将使用CNT阵列来提高蛋白质和核酸转移到细胞中的效率。 PSC。该假设是CNT装置表面的独特几何形状对于细胞活力和细胞生长都是至关重要的。 这意味着，CNT装置将有效地将DNA和蛋白质转移到PSC中。具体目标 （1）使用CRISPR递送的人PSC中的插入缺失突变的增强产生， （2）在PSC中开发CNT增强的同源定向重组（HDR）。我们将转移 将具有gRNA和HDR寡核苷酸的预包装的重组Cas9植入iPSC中。对于这些研究，我们将使用 含有内源性EGFP基因的iPSC，并通过荧光激活细胞监测编辑效率 分选（FACS）、荧光显微镜和EGFP等位基因的DNA测序。 这些研究将建立使用CNT阵列在PSC中进行CRISPR基因编辑的条件和效率， 核酸/蛋白质复合物的有效递送。未来的II期研究将扩展这一点，以开发器械 适用于有效的全基因组CRISPR筛选和快速生成用于疾病的同基因iPSC的形式 建模

ABSTRACT The goal of the proposed studies is to improve the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Currently, CRISPR/Cas9 shows great potential for targeted gene editing, but remains challenging in PSCs due to their being highly refractory to conventional transfection methods compared to other primary cell types and cell lines. This poses a significant hurdle to the targeted genetic manipulation of PSCs. What is needed is a transfection method that is fast, efficient, requires fewer input cells, and can introduce DNA/RNA/protein complexes into PSCs with minimal toxicity. AGTC has developed a novel method to efficiently introduce biomolecules into mammalian cells using devices composed of an array of closely packed and aligned carbon nanotubes (CNT) to achieve highly efficient transfer with low cytotoxicity. AGTC has also developed a scalable nanomanufacturing process for these CNT devices using template-based chemical vapor deposition (CVD) to produce a device consisting of thousands of 200 nm- diameter hollow carbon nanotubes (CNT) embedded in a 13 mm-diameter base which can be used with standard tissue culture plates. In this proposal, AGTC will use CNT arrays to increase the efficiency of transfer of protein and nucleic acids into PSCs. The hypothesis is that the unique geometry of the CNT device surface is critical to both cell viability and biomolecule transfer, and that CNT devices will efficiently transfer DNA and protein into PSCs. The specific aims of this Phase I proposal are: (1) Enhanced production of indel mutations in human PSCs using CRISPR delivered by CNT, and (2) Develop CNT-enhanced, homology-directed recombination (HDR) in PSCs. We will transfer prepackaged recombinant Cas9 with gRNA and HDR oligonucleotides into iPSCs. For these studies, we will use iPSCs that contain an endogenous EGFP gene, and monitor editing efficiency by fluorescence activated cell sorting (FACS), fluorescence microscopy, and DNA sequencing of the EGFP allele. These studies will establish the conditions and efficiency for CRISPR gene editing in PSCs using CNT arrays for efficient delivery of nucleic acid/protein complexes. Future Phase 2 studies will expand this to develop device formats suitable for efficient genome-wide CRISPR screens and rapid generation of syngeneic iPSCs for disease modeling.