Genome engineering with custom recombinases.
Site-specific recombinases are valuable tools for myriad basic research and genome engineering applications. In particular, hybrid recombinases consisting of catalytic domains from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are capable of introducing targeted modifications into mammalian cells. Due to their inherent modularity, new recombinases with distinct targeting specificities can readily be generated and utilized in a "plug-and-play" manner. In this protocol, we provide detailed, step-by-step instructions for generating new hybrid recombinases with user-defined specificity, as well as methods for achieving site-specific integration into targeted genomic loci using these systems.
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA; Department of Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA. Electronic address: gaj@berkeley.edu.
Thomas
T
Gaj
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA; Department of Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.
Carlos F
CF
Barbas
D000595:Amino Acid Sequence; D000818:Animals; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D016335:Zinc Fingers
D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase
Genome engineering; Protein engineering; Recombinase; TAL effector; Zinc-finger