title: Chimeric TALE recombinases with programmable DNA sequence specificity.
abstract: Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Mercer
authors.forename: Thomas
authors.initials: T
authors.lastname: Gaj
authors.forename: Roberta P
authors.initials: RP
authors.lastname: Fuller
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015723:Gene Library; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry* / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D004247:DNA; D004254:DNA Nucleotidyltransferases; C056830:DNA invertase Gin