Abstract
Single-strand nicking endonucleases (“nickases”) have been shown to induce homology-mediated gene correction with reduced toxicity of DNA double-strand break-producing enzymes, and nickases have been engineered from both homing endonuclease and FokI-based scaffolds. We describe the strategies used to engineer these site-specific nickases as well as the in vitro methods used to confirm their activity and specificity. Additionally, we describe the Traffic Light Reporter system, which uses a flow cytometric assay to simultaneously detect both gene repair and mutagenic nonhomologous end-joining outcomes at a single targeted site in mammalian cells. With these methods, novel nickases can be designed and tested for use in gene correction with novel target sites.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Certo MT, Ryu BY, Annis JE et al (2011) Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 8(8):671–676
Davis L, Maizels N (2011) DNA nicks promote efficient and safe targeted gene correction. PLoS One 6(9):e23981
McConnell SA, Takeuchi R, Pellenz S et al (2009) Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc Natl Acad Sci USA 106(13):5099–5104
Metzger MJ, McConnell-Smith A, Stoddard BL et al (2011) Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template. Nucleic Acids Res 39(3):926–935
Ramirez CL, Certo MT, Mussolino C et al (2012) Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Res 40(12):5560–5568
Mali P, Aach J, Stranges PB et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnol 31(9):833–838
Ran FA, Hsu PD, Lin CY et al (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389
Niu Y, Tenney K, Li H et al (2008) Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity. J Mol Biol 382(1):188–202
Kim E, Kim S, Kim DH et al (2012) Precision genome engineering with programmable DNA-nicking enzymes. Genome Res 22(7):1327–1333
Wang J, Friedman G, Doyon Y et al (2012) Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme. Genome Res 22(7):1316–1326
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823-826
Gabsalilow L, Schierling B, Friedhoff P et al (2013) Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats. Nucleic Acids Res 41(7):e83
Beurdeley M, Bietz F, Li J et al (2013) Compact designer TALENs for efficient genome engineering. Nat Commun 4:1762
Naldini L, Blomer U, Gage FH et al (1996) Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A 93(21):11382–11388
Takeuchi R, Certo M, Caprara MG et al (2009) Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation. Nucleic Acids Res 37(3):877–890
Cornea AM, Russell DW (2010) Chromosomal position effects on AAV-mediated gene targeting. Nucleic Acids Res 38(11):3582–3594
Acknowledgments
This work was supported by fellowships from National Institute of Health to MJM (CA009657 and CA009503) and MTC (GM007270). We thank Ryo Takeuchi for the purified I-AniIY2.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Metzger, M.J., Certo, M.T. (2014). Design and Analysis of Site-Specific Single-Strand Nicking Endonucleases for Gene Correction. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-761-7_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-760-0
Online ISBN: 978-1-62703-761-7
eBook Packages: Springer Protocols