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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, NY) 337(6096):816–821. https://doi.org/10.1126/science.1225829
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) Rna-guided human genome engineering via cas9. Science (New York, NY) 339(6121):823–826. https://doi.org/10.1126/science.1232033
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using crispr/cas systems. Science (New York, NY) 339(6121):819–823. https://doi.org/10.1126/science.1231143
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the crispr-cas9 system. Nat Protoc 8(11):2281–2308. https://doi.org/10.1038/nprot.2013.143
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of rna-guided cas9 nucleases. Nat Biotechnol 31(9):827–832. https://doi.org/10.1038/nbt.2647
Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by crispr-cas nucleases in human cells. Nat Biotechnol 31(9):822–826. https://doi.org/10.1038/nbt.2623
Moore CBT, Christie KA, Marshall J, Nesbit MA (2018) Personalised genome editing - the future for corneal dystrophies. Prog Retin Eye Res 65:147–165. https://doi.org/10.1016/j.preteyeres.2018.01.004
Christie KA, Courtney DG, DeDionisio LA, Shern CC, De Majumdar S, Mairs LC, Nesbit MA, Moore CBT (2017) Towards personalised allele-specific crispr gene editing to treat autosomal dominant disorders. Sci Rep 7(1):16174. https://doi.org/10.1038/s41598-017-16279-4
Courtney DG, Moore JE, Atkinson SD, Maurizi E, Allen EH, Pedrioli DM, McLean WH, Nesbit MA, Moore CB (2016) Crispr/cas9 DNA cleavage at snp-derived pam enables both in vitro and in vivo krt12 mutation-specific targeting. Gene Ther 23(1):108–112. https://doi.org/10.1038/gt.2015.82
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Moore, T., Chao-Shern, C., DeDionisio, L., Christie, K.A., Nesbit, M.A. (2020). Gene Editing for Corneal Stromal Regeneration. In: Ahearne, M. (eds) Corneal Regeneration. Methods in Molecular Biology, vol 2145. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0599-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0599-8_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0598-1
Online ISBN: 978-1-0716-0599-8
eBook Packages: Springer Protocols