title: Engineering of customized meganucleases via in vitro compartmentalization and in cellulo optimization.
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.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., A3-025, Seattle, WA, 98109-1024, USA.
authors.forename: Ryo
authors.initials: R
authors.lastname: Takeuchi
authors.forename: Michael
authors.initials: M
authors.lastname: Choi
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*
chemical_list: D004720:Endonucleases; D004262:DNA Restriction Enzymes