Abstract
LAGLIDADG homing endonucleases (also referred to as “meganucleases”) are compact DNA cleaving enzymes that specifically recognize long target sequences (approximately 20 base pairs), and thus serve as useful tools for therapeutic genome engineering. While stand-alone meganucleases are sufficiently active to introduce targeted genome modification, they can be fused to additional sequence-specific DNA binding domains in order to improve their performance in target cells. In this chapter, we describe an approach to retarget meganucleases to DNA targets of interest (such as sequences found in genes and cis regulatory regions), which is feasible in an academic laboratory environment. A combination of two selection systems, in vitro compartmentalization and two-plasmid cleavage assay in bacteria, allow for efficient engineering of meganucleases that specifically cleave a wide variety of DNA sequences.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kolb AF, Coates CJ, Kaminski JM, Summers JB, Miller AD, Segal DJ (2005) Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction. Trends Biotechnol 23:399–406
Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–782
Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646
Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846
Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761
Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39:359–372
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
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 339:823–826
Arnould S, Perez C, Cabaniols JP, Smith J, Gouble A, Grizot S, Epinat JC, Duclert A, Duchateau P, Paques F (2007) Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol 371:49–65
Stoddard BL (2011) Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification. Structure 19:7–15
Segal DJ, Meckler JF (2013) Genome engineering at the dawn of the golden age. Annu Rev Genomics Hum Genet 14:135–158
Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, Paques F (2011) Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther 11:11–27
Brunet E, Simsek D, Tomishima M, DeKelver R, Choi VM, Gregory P, Urnov F, Weinstock DM, Jasin M (2009) Chromosomal translocations induced at specified loci in human stem cells. Proc Natl Acad Sci U S A 106:10620–10625
Lee HJ, Kweon J, Kim E, Kim S, Kim JS (2012) Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res 22:539–548
Sollu C, Pars K, Cornu TI, Thibodeau-Beganny S, Maeder ML, Joung JK, Heilbronn R, Cathomen T (2010) Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion. Nucleic Acids Res 38:8269–8276
Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys 38:49–95
Certo MT, Ryu BY, Annis JE, Garibov M, Jarjour J, Rawlings DJ, Scharenberg AM (2011) Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods 8:671–676
Daboussi F, Zaslavskiy M, Poirot L, Loperfido M, Gouble A, Guyot V, Leduc S, Galetto R, Grizot S, Oficjalska D et al (2012) Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res 40:6367–6379
Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, West A, Bidney D, Falco SC, Jantz D et al (2010) Heritable targeted mutagenesis in maize using a designed endonuclease. Plant J 61:176–187
Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G et al (2009) Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res 37:5405–5419
Takeuchi R, Lambert AR, Mak AN, Jacoby K, Dickson RJ, Gloor GB, Scharenberg AM, Edgell DR, Stoddard BL (2011) Tapping natural reservoirs of homing endonucleases for targeted gene modification. Proc Natl Acad Sci U S A 108:13077–13082
Boissel SJ, Astrakhan A, Jarjour J, Adey A, Shendure J, Stoddard BL, Certo MT, Baker D, Scharenberg AM (2014) MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 42:2591
Takeuchi R, Choi M, Stoddard BL (2014) Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization. PNAS USA 111: 4061–4066
Miller OJ, Bernath K, Agresti JJ, Amitai G, Kelly BT, Mastrobattista E, Taly V, Magdassi S, Tawfik DS, Griffiths AD (2006) Directed evolution by in vitro compartmentalization. Nat Methods 3:561–570
Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656
Zheng Y, Roberts RJ (2007) Selection of restriction endonucleases using artificial cells. Nucleic Acids Res 35:e83
Doyon JB, Pattanayak V, Meyer CB, Liu DR (2006) Directed evolution and substrate specificity profile of homing endonuclease I-SceI. J Am Chem Soc 128:2477–2484
Taylor GK, Petrucci LH, Lambert AR, Baxter SK, Jarjour J, Stoddard BL (2012) LAHEDES: the LAGLIDADG homing endonuclease database and engineering server. Nucleic Acids Res 40:W110
Molina R, Redondo P, Stella S, Marenchino M, D’Abramo M, Gervasio FL, Epinat JC, Valton J, Grizot S, Duchateau P et al (2012) Non-specific protein-DNA interactions control I-CreI target binding and cleavage. Nucleic Acids Res 40:6936–6945
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Takeuchi, R., Choi, M., Stoddard, B.L. (2015). Engineering of Customized Meganucleases via In Vitro Compartmentalization and In Cellulo Optimization. In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1862-1_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1861-4
Online ISBN: 978-1-4939-1862-1
eBook Packages: Springer Protocols