Gene Editing for Corneal Stromal Regeneration

Tara C. B. Moore, Connie Chao-Shern, Larry DeDionisio, Kathleen A. Christie, M. Andrew Nesbit

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.
Original languageEnglish
Title of host publicationCorneal Regeneration
Subtitle of host publicationMethods in Molecular Biology
EditorsMark Ahearne
Place of PublicationNew York, NY
PublisherSpringer Nature
Chapter6
Pages59-75
Number of pages17
Volume2145
ISBN (Electronic)978-1-0716-0599-8
ISBN (Print)978-1-0716-0598-1
DOIs
Publication statusE-pub ahead of print - 16 Jun 2020

Publication series

NameMethods in Molecular Biology
Volume2145
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Gene editing
  • sgRNA expression construct
  • Dual luciferase assay
  • In vitro digest
  • Lymphoblastoid cell line
  • Nucleofection
  • Polymerase chain reaction (PCR)
  • Corneal endothelial and epithelial cell culture
  • In vivo imaging
  • Intracameral injection
  • Adeno-associated virus (AAV)

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  • Cite this

    Moore, T. C. B., Chao-Shern, C., DeDionisio, L., Christie, K. A., & Nesbit, M. A. (2020). Gene Editing for Corneal Stromal Regeneration. In M. Ahearne (Ed.), Corneal Regeneration: Methods in Molecular Biology (Vol. 2145, pp. 59-75). (Methods in Molecular Biology; Vol. 2145). Springer Nature. https://doi.org/10.1007/978-1-0716-0599-8_6