Abstract
Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.
Highlights
The precise manipulation of DNA is arguably one of the key technologies that hallmark the rapid progress in biomedical research
Novel approaches have been successfully developed to expand the variety of DNA-cleaving enzymes, with zinc-finger nucleases (ZFNs), TAL enhancer nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR) - associated nuclease (Cas) (CRISPR/Cas[9] system) revolutionizing the field of genome engineering[3]
The recently identified Vika recombinase, isolated from the gram-negative bacterium V. coralliilyticus recombines its 34-bp target site, vox, with high efficiency and specificity[23]. These enzymes expand the repertoire of available enzymes that are useful for advanced genome engineering and detailed characterization of these enzymes provides guidelines for their applied use
Summary
The precise manipulation of DNA is arguably one of the key technologies that hallmark the rapid progress in biomedical research. For a more guided approach and to accelerate the directed evolution process, it is desirable to understand how these enzymes bind and recombine their targets in a specific manner[20] These examples explain the growing interest in the identification of new naturally occurring SSR systems. The recently identified Vika recombinase, isolated from the gram-negative bacterium V. coralliilyticus recombines its 34-bp target site, vox, with high efficiency and specificity[23]. These enzymes expand the repertoire of available enzymes that are useful for advanced genome engineering and detailed characterization of these enzymes provides guidelines for their applied use. The identification of these, in combination with the here described novel SSR systems Nigri/nox and Panto/pox will allow the design of more sophisticated genome engineering experiments and advance our understanding on how these enzymes bind their DNA substrates
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