The goal of the proposed studies is to improve the efficiency of clustered regularly interspaced short palindromicrepeats (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 targetedgene editing, but remains challenging in PSCs due to their being highly refractory to conventional transfectionmethods compared to other primary cell types and cell lines. This poses a significant hurdle to the targetedgenetic manipulation of PSCs. What is needed is a transfection method that is fast, efficient, requires fewer inputcells, 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 devicescomposed of an array of closely packed and aligned carbon nanotubes (CNT) to achieve highly efficient transferwith low cytotoxicity. AGTC has also developed a scalable nanomanufacturing process for these CNT devicesusing 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 standardtissue culture plates.In this proposal, AGTC will use CNT arrays to increase the efficiency of transfer of protein and nucleic acids intoPSCs. The hypothesis is that the unique geometry of the CNT device surface is critical to both cell viability andbiomolecule transfer, and that CNT devices will efficiently transfer DNA and protein into PSCs. The specific aimsof this Phase I proposal are: (1) Enhanced production of indel mutations in human PSCs using CRISPR deliveredby CNT, and (2) Develop CNT-enhanced, homology-directed recombination (HDR) in PSCs. We will transferprepackaged recombinant Cas9 with gRNA and HDR oligonucleotides into iPSCs. For these studies, we will useiPSCs that contain an endogenous EGFP gene, and monitor editing efficiency by fluorescence activated cellsorting (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 forefficient delivery of nucleic acid/protein complexes. Future Phase 2 studies will expand this to develop deviceformats suitable for efficient genome-wide CRISPR screens and rapid generation of syngeneic iPSCs for diseasemodeling.
Public Health Relevance Statement: NARRATIVE
CRISPR (clustered regularly interspaced short palindromic repeats) gene editing is revolutionizing the field of
personalized medicine for a multitude of diseases; however, it is currently limited by inefficient transfer
techniques with low genetic load capabilities. In this proposal, we will use our proprietary carbon nanotube
technology (CNT) to improve the efficiency of CRISPR-based gene editing in human cells to reduce costs and
enable new screens for elucidating the causal mutations in disease.
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