Site-specific recombination (SSR) systems like Cre/lox and FLP/FRT allow for precise integration of genes of interest (GOI) into plant genomes compared to traditional transformation methods. The Cre/lox system uses the Cre recombinase to catalyze recombination between two loxP sites, while the FLP/FRT system uses FLP recombinase to catalyze recombination between two FRT sites. These systems allow for single-copy, single-locus integration of GOIs without disrupting functional genes. However, constitutive expression of Cre and FLP recombinases can cause unwanted genomic changes. Current research focuses on engineering these recombinases for improved efficiency and versatility of SSR for transgenic plant

1. SITE SPECIFIC RECOMBINATION
(SSR)

2. WHY SSR???
• Transformation through Agrobacterium & direct
DNA transfer leads to complex integration of
GOI.
• Complex integration is classified into,
 Single copy – multi locus integration
 Multi copy – single locus integration
• Complex integration leads to gene silencing.

3. • Ideal integration is single copy – single locus and
integration of GOI without intervening with
functional gene.
• The best way out of this issue is promoting the
development of system/technique that facilitates
site specific recombination.

4. • Two systems have been developed for
facilitating site specific recombination,
namely,
Cre/lox system
FLP/FRT system

5. Cre/lox system
• Derived from P1 bacteriophage.
• Cre recombinase catalyses the recombination
between two loxP(site for recombination) sites.
• loxP site consists of an 8-bp core sequence, where
recombination takes place, and two flanking 13-bp
inverted repeats.(In total 34-bp)

6. loxP site

7. The Jackson Laboratory

8. FLP/FRT system
• Derived from 2 micrometer yeast plasmid
• FLP recombinase enhances recombination of
sequences between two short Flippase
Recognition Target (FRT).
• FRT consists of an 8-bp spacer and two flanking
13-bp inverted repeats, where recombination
takes place (In total 34-bp).

9. FRT site

10. Classification based on nature of
Recombination
• Recombinase Mediated Cassette Exchange(RMCE)
strategy:
Based on dual recombination between a
pair of recombination sites resulting in
exchange of DNA cassettes between the
target locus and the donor DNA.

12. • Co-integration strategy:
In this method, single recombination
between two recombination sites occur,
resulting in integration of circular DNA
into the target site.

13. Plant Biotechnology Journal

14. • Site specific recombination – silver bullet for
enhancing ideal gene expression.
• Despite several advantages there are certain
limitations,
 Constitutive expression of Cre , FLP
protein is a problem- leads to undesired
genotypic changes.
Transfer of cre, flp gene to the progeny
lines is challenging.
The efficiency of these systems are not
up to the mark and they are not versatile.

15. CURRENT STATUS
• In order enhance the efficiency of site specific
recombination system(enzymes),
The current research focuses on the
alteration of the catalytic properties to
in turn modify the characters like
thermostability, recognition
sequences etc…

16. REFERENCES
 Site specific gene integration in rice genome mediated by
FLP-FRT recombination system. Soumen Nandy , Vibha
Srivastava , Plant Biotechnology Journal (2011) 9, pp. 713–
721
 Elimination of transgenic sequences in plants by cre gene
expression.
Book : Transgenic Plants-Advances & limitations,
Authors: Lilya Kopertekh and Joachim Schiemann
 FLP recombinase mediated site-specific recombination in
rice. Qian Hu , Halina Kononowicz-Hodges, Kimberly Nelson-
Vasilchik, David Viola, Peiyu Zeng, Haibo Liu, Albert P. Kausch ,
Joel M. Chandlee, Thomas K. Hodges and Hong Luo. Plant
Biotechnology Journal(2008)6, pp. 176–188

17.  An ultrasensitive site-specific DNA
recombination assay based on dual-color
fluorescence cross-correlation
spectroscopy.Michael Jahnz and Petra Schwille.
Nucleic Acids Research, 2005, Vol. 33, No. 6 e60
www.wikipedia.com