Team:Edinburgh UG/Description

Project Description

What is site-specific recombination and why does it matter?

Site-specific recombination (SSR), found mostly in bacteria, viruses and transposons (the so-called parasitic DNA), is one of the many mechanisms which life utilises to perform genetic recombination (Leach, 1996). SSR generally consists of a recombinase protein that mediates recombination, and two DNA elements, called target sites, that are similar or identical to each other that the recombinase recognises (Grindley et al., 2006). Depending on the orientation and the location of the target sites, SSR can perform DNA integration, excision, and exchange. While bacteria use SSR to regulate gene expression and separate two chromosomes during cell division, viruses and transposons use it to mediate chromosomal integration into the bacterial chromosome, hijacking the cellular machinery to replicate themselves.

With the ability to modify DNA in a precise manner, SSR has been used in various fields of research and industrial applications where genetic engineering is required. Click to see the applications of SSR:

Genetic Engineering

a. Conditional Gene Knockout

Site-specific recombinases are highly efficient in excising DNA segments flanked with target sites; this property has been widely adopted by neuroscientists and developmental biologists to perform conditional gene knockout, where a gene can be deleted in specific time and cell types, and is particularly useful to study genes that are essential for development. For example, Hara et al. showed that autophagy was important not only for adaptation to starvation, but also for preventing degeneration of neurons. They demonstrated it by knocking out Atg5, a protein involved in autophagy, specifically in neurons using Cre/LoxP recombination system, as its complete knockout is lethal to the organism.

Examples:
(1) Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y., Suzuki-Migishima, R., Yokoyama, M., Mishima, K., Saito, I., Okano, H., Mizushima, N. (2006) Suppression of autophagy in neural cells causes neurodegenerative disease in mice. Nature. 441: 885-889
(2) Yang, X., Li, C., Herrera, P., Deng, C. (2002) Generation of Smad4/Dpc4 conditional knockout mice. Genesis. 32(2): 80-81

b. Transgenesis

SSR has been very useful for inserting genes into designated loci of a genome. This is more advantageous than strategies based on homologous recombination (HR), in that 1) it is more efficient , 2) concatenation effect [insert citation here] can be eliminated, and 3) selective marker can be removed with ease. Such properties have been exploited into advanced techniques such as recombinase-mediated cassette exchange (RMCE) and serine integrase recombinational assembly (SIRA), where multiple genes can be introduced and exchanged rapidly. They often complement with other genetic engineering tools (e.g. CRISPR-Cas system) to perform desired functions.

Examples:
(1) Bischof, J., Maeda, R.A., Hediger, M., Karch, F., Basler, K. (2006) An optimised transgenesis system for Drosophila using germline-specific φC31 integrases. Proc Natl Acad Sci. 104(9): 3312-3317
(2) Hammond, A., Galizi, R., Kyrou,K., Simoni, A., Siniscalchi, C., Katsanos, D., Gribble, M., Baker, D., Marois, E., Russell, S., Burt, A., Windbichler, N., Crisanti, A., Nolan, T. (2016) A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nature Biotechnology. 34(1): 78-83
(3) Colloms, S.D., Merrick, C.A., Olorunniji, F.J., Stark, W.M., Smith, M.C.M., Osbourn, A., Keasling, J.D., Rosser, S.J. (2014) Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Res. 42(4): e23

c. Large-scale chromosome modification

Although being gradually replaced by the more recent technology such as CRISPR/Cas system, SSR has been employed to perform large scale DNA modifications (deletion, insertion, and inversion), and is a handy tool for modeling diseases that are caused by chromosome rearrangements. Compared to CRISPR technology, SSR does not rely on the organism’s ability to repair double-stranded breaks (DSBs) by homologous recombination, and is therefore particularly useful in organisms such as bacteria that have limited ability to repair DSBs.

Examples:
(1) Venken, , K.J., He, Y., Hoskins, R.A., Bellen, H.J. (2006) P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science. 314(5806): 1747-51
(2) Uemura, M., Niwa, Y., Kakazu, N., Adachi, N., Kinoshita, K. (2010) Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors. PLoS ONE 5(3): e9846
(3) Enyeart, P.J., Chirieleison, S.M., Dao, M.N., Perutka, J., Quandt, E.M., Yao, J., Whitt, J.T., Keatinge-Clay, A.T., Lambowitz, A.M., Ellington, A.D. (2013) Generalized bacterial genome editing using mobile group II introns and Cre-lox. Molecular Systems Biology. 9(1): 685

Research

a. Disease modeling

SSR, as an genetic engineering tool, is widely used to simulate and study pathological conditions such as inactivational mutations and chromosomal rearrangements. For example, Barlow et al. used Cre/LoxP mechanism to delete a part of the mouse chromosome 18 to study the 5q- syndrome in human, and they found that the deletion was associated with a change in p53 level, providing insight on its disease mechanism.

Examples:
(1) Barlow, J.L., Drynan, L.F., Hewett, D.R., Holmes, L.R., Lorenzo-Abalde, S., Lane, A.L., Jolin, H.E., Pannell, R., Middleton, A.J., Wong, S.H., Warren, A.J., Wainscoat, J.S., Boultwood, J., McKenzie, A.N.J. (2010) A p53-dependent mechanism underlies macrolytic anemia in a mouse model of human 5q-syndrome. Nature medicine. 16: 59-66
Pan, L., Wang, S., Lu, T., Weng, C., Song, X., Park, J.K., Sun, J., Yang, Z-H., Yu, J., Tang, H., McKearin, D.M., Chamovitz, D.A., Ni, J., Xie, T. (2014) Protein competition switches the function of COP9 from self-renewal to differentiation. Nature. 514(7521): 233-6

b. Cell lineage tracing

Lineage tracing is the tagging of a specific population of cells that allows researchers to study their role in development. This has been very useful in not only the identification of stem cells, but also in the validation of the cancer stem cells theory. Furthermore, Cre/LoxP recombination system has been implemented in the Brainbow system, where more than 100 individual neuron cells can be distinguished from each other in the same image. This unprecedented technique has shone light to the study of connectome, the study of architecture and interaction between neurons in a brain.

Examples:
(1) Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., Clevers, H. (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 449: 1003-1007
Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., Lichtman, J.W. (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature. 450: 56-62
(3) Pei, W., Feyerabend, T.B., Rössler, J., Wang, X., Postrach, D., Busch, K., Rode, I., Klapproth, K, Dietlein, N., Quadenau, C., Chen, W., Sauer, S., Wolf, S., Höfer, T., Rodewald, H. (2017) Polylox barcoding reveals haematopoietic stem cell fates realized in vivo. Nature. 548(7668): 456-460

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