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

使用碳纳米管阵列增强多能干细胞中的 CRISPR 基因编辑

基本信息

批准号：
10698269
负责人：
IAN M DICKERSON
金额：
$ 27.5万
依托单位：
ADVANCED GENE TRANSFER COMPANY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-05 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10698269
关键词：
Actins Alleles Benchmarking Buffers CRISPR screen Carbon Nanotubes Cell Line Cell Survival Cell model Cells Chemicals Chimeric Proteins Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Complex DNA DNA sequencing Deposition Devices Diameter Disease Disease model Fluorescence Fluorescence Microscopy Fluorescence-Activated Cell Sorting Future Gene Expression Gene Fusion Gene Proteins Generations Genes Genetic Genetic Load Genetic Recombination Genetic Screening Geometry Goals Guide RNA Human Libraries Mammalian Cell Mediating Methods Monitor Mutation Nanomanufacturing Nanotubes Nucleic Acids Nucleotides Oligonucleotides Patients Phase Pluripotent Stem Cells Point Mutation Production Property Proteins RNA Recombinants Refractory Reporter Site Surface Techniques Technology Therapeutic Toxic effect Transfection base causal variant cell type cost culture plates cytotoxicity design embryonic stem cell enhanced green fluorescent protein experimental study gain of function genetic manipulation genome editing genome-wide human pluripotent stem cell improved induced pluripotent stem cell insertion/deletion mutation lipofection loss of function mutation manufacture manufacturing process novel nucleic acid delivery personalized medicine phase 1 study phase 2 study protein complex protein function red fluorescent protein repaired tissue culture vapor

项目摘要

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.

抽象的所提出研究的目标是提高聚类规则间隔短回文的效率对人类多能干细胞 (PSC)（包括胚胎干细胞）进行基于重复 (CRISPR) 的基因编辑 (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，但由于 PSC 对传统转染非常难治，因此在 PSC 中仍然具有挑战性与其他原代细胞类型和细胞系相比的方法。这对目标实现构成了重大障碍 PSC 的基因操作。我们需要一种快速、高效、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 开发了一种使用设备有效地将生物分子引入哺乳动物细胞的新方法由一系列紧密排列的碳纳米管（CNT）组成，可实现高效转移具有低细胞毒性。 AGTC 还为这些 CNT 设备开发了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- 直径空心碳纳米管 (CNT) 嵌入直径 13 毫米的底座中，可与标准一起使用组织培养板。在该提案中，AGTC 将使用 CNT 阵列来提高将蛋白质和核酸转移到 PSC。假设 CNT 器件表面的独特几何形状对于细胞活力和生物分子转移，CNT 装置将有效地将 DNA 和蛋白质转移到 PSC 中。具体目标该第一阶段提案的内容是：(1) 使用 CRISPR 增强人类 PSC 中 indel 突变的产生 (2) 在 PSC 中开发 CNT 增强的同源定向重组 (HDR)。我们将转移将带有 gRNA 和 HDR 寡核苷酸的重组 Cas9 预包装到 iPSC 中。对于这些研究，我们将使用含有内源 EGFP 基因的 iPSC，通过荧光激活细胞监测编辑效率 EGFP 等位基因的分选 (FACS)、荧光显微镜和 DNA 测序。这些研究将确定使用 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。未来的第二阶段研究将扩大这一范围以开发设备适合高效全基因组 CRISPR 筛选和快速生成疾病同基因 iPSC 的格式造型。