2. Academic Validation
  3. Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion

Supramolecular Nanosubstrate-Mediated Delivery for CRISPR/Cas9 Gene Disruption and Deletion

  • Small. 2021 Jul;17(28):e2100546. doi: 10.1002/smll.202100546.
Qian Ban 1,  Peng Yang 2,  Shih-Jie Chou 3,  Li Qiao 1,  Haidong Xia 1,  Jingjing Xue 2,  Fang Wang 4,  Xiaobin Xu 5 6,  Na Sun 2,  Ryan Y Zhang 2,  Ceng Zhang 2,  Athena Lee 2,  Wenfei Liu 5,  Ting-Yi Lin 3,  Yu-Ling Ko 3,  Petar Antovski 2,  Xinyue Zhang 2,  Shih-Hwa Chiou 3,  Chin-Fa Lee 7,  Wenqiao Hui 8,  Dahai Liu 9,  Steven J Jonas 10,  Paul S Weiss 5,  Hsian-Rong Tseng 2
Affiliations

Affiliations

  • 1 School of Life Sciences, Center for Stem Cell and Translational Medicine, Anhui University, Hefei, 230601, China.
  • 2 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, California NanoSystems Institute (CNSI), Crump Institute for Molecular Imaging (CIMI), University of California, Los Angeles, Los Angeles, CA, 90095, USA.
  • 3 Department of Medical Research, and Stem Cell Center, Division of Basic Research, Taipei Veterans General Hospital, Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, No. 155, Sec. 2, Linong Street 112, Taipei, Taiwan.
  • 4 Department of Macromolecular Science, State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai, 200433, China.
  • 5 Department of Chemistry and Biochemistry, Department of Bioengineering, Department of Materials Science and Engineering, California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA, 90095, USA.
  • 6 School of Materials Science and Engineering, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
  • 7 Department of Chemistry, i-Center for Advanced Science and Technology (iCAST), Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University (NCHU), 145 Xingda Road, South Dist., Taichung, 402, Taiwan.
  • 8 Anhui Province Key Laboratory of Livestock and Poultry Product Safety Engineering, Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agriculture Sciences, Hefei, 230031, China.
  • 9 School of Stomatology and Medicine, Foshan University, Foshan, 528000, China.
  • 10 Department of Pediatrics, David Geffen School of Medicine, California NanoSystems Institute (CNSI), Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Children's Discovery and Innovation Institute, University of California, Los Angeles, Los Angeles, CA, 90095, USA.
PMID: 34105245 DOI: 10.1002/smll.202100546
Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green Fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

Keywords

CRISPR/Cas9; Duchenne muscular dystrophy; gene editing; nanosubstrate-mediated delivery; supramolecular nanoparticles.

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