Site-specific recombinases

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Site-specific DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes and, in turn, to control gene expression (for reviews see refs below). These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30–40 nucleotides) target site sequences that are specific to each recombinase [1]. These reactions enable four basic functional modules—excision/insertion, inversion, translocation and cassette exchange—that have been used individually or combined in a wide range of configurations to control gene expression (Fig. 1).

Cre recombinase

Fig. 1. Modifying genome structure and gene expression with site-specific recombinases. (A) Four types of reaction are diagramed. The target sites recognized by the recombinases are indicated by the colored triangles, and the black lines represent genomic DNA. In the excision/insertion reaction, a segment of DNA between two tandemly arranged target sites can be excised as a circular molecule. The reverse reaction, insertion, occurs with much lower efficiency. In the inversion reaction, a segment of DNA between two oppositely oriented target sites can undergo one or more cycles of inversion. In the translocation reaction, a segment of a chromosome arm distal to the centromere is exchanged between homologs in a diploid organism. In the cassette exchange reaction, a linear segment of DNA is exchanged between two DNA molecules. (B) An example of the use of two recombinases with distinct target sites (represented by the different colored triangles) to perform a genetic intersection (2). From [2].

Cre recombinase, like all site-specific recombinases (SSRs), excises or recombines DNA depending on the relative orientation of short, directional DNA sequences [3]. The 34 base pair (bp) lox sites, recognized by Cre, consist of two 13 bp palindromic regions and an intervening non-palindromic 8 bp spacer that determines the orientation of the site. When two lox sites are oriented in the same direction, Cre excises the DNA flanked by the lox sites, leaving a single lox site behind. Conversely, when the lox sites are oriented in the opposite direction, Cre flips the flanked DNA into the antisense orientation. Both reactions involve the exchange of DNA between the two lox sites and are normally reversible [4].

Differences in palindromic or spacer regions of lox sites, either naturally occurring [5] or randomly mutated [6], can confer specificity to Cre recognition. Exploiting lox variants that undergo variant-specific recombination has enabled strategies for making Cre recombination effectively irreversible [7, 8, 9, 10]. The FLEx system, first used as a Cre-reporter (Schnütgen et al. 2003) and then applied to rAAV transgenes [10, 11], uses recombination between two pairs of like loxP and lox2272 sites to confer a permanent recombination event. Expression in the presence of Cre (“Cre-On”) is achieved by FLEx recombination of a transgene that changes the orientation of the coding sequence with respect to the promoter from the anti-sense to sense. Conversely, inactivation of expression in the presence of Cre (“Cre-Off”) can be achieved by simply starting the transgene in the sense orientation.

Cre-On rAAVs have been used widely to study the function of Cre-expressing neuron populations ([12, 13] and Cardin et al. 2009), whereas Cre-Off rAAVs, despite their experimental value, have received only minor attention [10, 14].

Lox sites

  • LoxP
  • Lox2272
  • Lox FAS [5].

Flp recombinase

Flp is the most widely used recombinase in Drosophila, encoded by the Saccharomyces cerevisiae 2-μm plasmid [15]. Flp was first shown to work in a heterologous, multicellular organism by Golic and Lindquist in 1989 [16] who demonstrated the excision reaction on chromosomally inserted target sites (FRTs). Since that time FLP/FRT recombination has been widely used in Drosophila in applications based on excision [17] and translocation (Golic 1991) [18, 19].

Other recombinases

See for example: Nern et al., 2011. Multiple new site-specific recombinases for use in manipulating animal genomes, Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14198-203. [2]. Nern et al tested 4 new recombinases derived from yeast: KD, B2, B3, and R.

Specific applications

FLEx

Brainbow

Brainbow 1.1
Legend here.
Legend here.

Links

Reviews

References

Text above taken from [21] and [2].

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  1. Error fetching PMID 16756503: [Grindley2006]
  2. Error fetching PMID 21831835: [Nern2011]
  3. Error fetching PMID 12354622: [Tronche2002]
  4. Error fetching PMID 11340053: [VanDuyne2001]
  5. Error fetching PMID 11576551: [Siegel2001]
  6. Error fetching PMID 17702764: [Sheren2007]
  7. Error fetching PMID 7742860: [Albert1995]
  8. Error fetching PMID 9016639: [Araki1997]
  9. Error fetching PMID 18614669: [Atasoy2008]
  10. Error fetching PMID 19396159: [Sohal2009]
  11. Error fetching PMID 20613723: [Kravitz2010]
  12. Error fetching PMID 22258508: [Cohen2012]
  13. Error fetching PMID 22138823: [Kim2011]
  14. Error fetching PMID 6251374: [Hartley1980]
  15. Error fetching PMID 2509077: [Golic1989]
  16. Error fetching PMID 8440019: [Struhl1993]
  17. Error fetching PMID 8404527: [Xu1993]
  18. Error fetching PMID 10197526: [Lee1999]
  19. Error fetching PMID 17972876: [Livet2007]
  20. Error fetching PMID 22866029: [Saunders2012]
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