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

点击翻译按钮获取中文摘要

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.