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
(2) 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
(2) 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

Synthetic Biology

a. Complex genetic circuits

Having the ability to perform both irreversible and reversible DNA modification, SSR has been used to create time delay circuits, logic gates, state machines and more. These have found applications on creating complex biosensors -- the detection of specific chemicals using biological pathways. Examples include the detection of multiple gaseous chemicals and integrating the signals using AND, OR, and NOR gates, and the sensitive detection of intracellular miRNA level for developmental biology research.

Examples:
(1) Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S., Lu, T.K. (2016) Synthetic recombinase-based state machines in living cells. Science. 353(6297): aad8559
(2) Weinberg, B.H., Hang Pham, N.T., Caraballo, L.D., Lozanoski, T., Engel, A., Bhatia, S., Wong, W.W. (2017) Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nature biotechnology. 35(5): 453-462
(3) Müller, M., Ausländer, S., Spinnler, A., Ausländer, D., Sikorski, J., Folcher, M., Fussenegger, M. (2017) Designed cell consortia as fragrance-programmable analog-to-digital converters. Nature chemical biology. 13: 309-316
(4) Lapique, N., Benenson, Y. (2014) Digital switching in a biosensor circuit via programmable timing of gene availability. Nature chemical biology. 10: 1020-1027

b. Directed evolution

Evolution is the change in the genetic content of a population of organisms through processes such as mutagenesis and natural selection. The 4.5 billion years of evolution have allowed organisms to diversify and generate numerous traits or combinations of traits; however, evolution itself is a slow process, and sometimes desirable traits may not be easily found in nature. This could be sped up by direction evolution, a method to mimic the process of evolution in an artificial way. Although there are many established methods of directed evolution, one of the most extraordinary technology is SCRaMbLE, which generated synthetic chromosome in yeast and in that, multiple recombinations sites were included. In the presence of recombinase, the sequences in the synthetic will go through random inversion, deletion, or duplication. This could potentially be apply not only to screen for the yeast strains with the best performance for a particular application, but also to facilitate research in the minimal genome. It is envisaged that similar system could be applied to various model organisms to facilitate research.

Examples:
(1) Dymond, J.S., Richardson, S.M., Coombes, C.E., Babatz, T., Müller, H., Annaluru, N., Blake, W.J., Schwerzmann, J.W., Dai, J., Lindstrom, D.L., Boeke, A.C., Gottschling, D.E., Chandrasegaran, S., Bader, J.S., Booke, J.D. (2011) Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature. 477(7365): 471-476
(2) Shen, Y., Stracquadanio, G., Wang, Y., Yang, K., Mitchell, L.A., Xue, Y., Cai, Y., Chen, T., Dymond, J.S., Kang, K., Gong, J., Zeng, X., Zhang, Y., Li, Y., Feng, Q., Xu, X., Wang, J., Wang, J., Yang, H., Booke, J.D., Bader, J.S. (2016) SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes. Genome research. 26: 36-49

Medical application

a. Diagnostics

Accurate diagnosis plays a decisive role in effective disease treatment. Therefore, much of the research effort is put towards its improvement. Particularly, recent years have seen a growing interest in the in vivo diagnostics. Using SSR and synthetic biology, researchers have engineered bacteria that normally inhabit human gut into surveillance machines that sense the gut environment and microbiota. They could potentially be used to record a patient’s past exposure of infections, informing clinicians to prescribe more appropriate therapy.

Examples:
(1) Rubens, J.R., Selvaggio, G., Lu, T.K. (2016) Synthetic mixed-signal computation in living cells. Nature communications. 7:11658
(2) Mimee, M., Tucker, A.C., Voigt, C.A., Lu, T.K. (2015) Programming a human commensal bacterium, Bacteroides thetaiotaomicron, to sense and respond to stimuli in murine gut microbiota. Cell systems. 1:62-71

b. Gene therapy

Gene therapy holds promise in curing diseases previously thought to be impossible to treat. One of the many challenges it faces is the need to lengthen the gene expression time while avoiding its permanent integration into the host genome. A prime example is the use of gene therapy to temporarily stimulate osteogenesis in stem cells, thereby healing bone defects. To enhance the effect of gene therapy, researchers use SSR to circularize the gene fragments transfected into the cells. As circular DNA is less subject to DNA degradation than does linear DNA, this method allows the gene to be expressed for a longer period, enhancing the healing potential of the stem cells. Also, SSR is being investigated to cure HIV, and similar diseases such as T-cell leukemia that are caused by retrovirus.

Examples:
(1) Lo, S., Li, K., Chang, Y., Hsu, M., Sung, L., Vu, T. A., Hu, Y. (2017) Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus. Biomaterial. 124: 1-11
(2) Sarker, I., Hauber, I., Hauber, J., Buchholz, F. (2007) HIV-1 proviral DNA excision using an evolved recombinase. Science. 316(5833): 1912-1915

c. Regenerative medicine

The discovery of the formula -- the so-called Yamanaka’s cocktail -- to generate induced pluripotent stem cells (iPSCs) has opened up myriad possibilities for regenerative medicine, a branch of research that deals with treating diseases through stimulating organs to heal themselves, or to grow organs and tissues in vitro from sources such as stem cells. However, the process of creating iPSCs involves introducing several potential oncogenes into the somatic cells, causing concerns over its safety in clinical use. Fortunately, these transgenes can be removed efficiently using SSR, and this technique has been widely adopted to treat diseases such as Duchenne muscular dystrophy (DMD) and 𝛽-thalassemia.

Examples:
(1) Zhao, C., Farruggio, A.P., Bjornson, C.R.R., Chavez, C.L., Geisinger, J.M., Neal, T.L., Karow, M., Calos, M.P. (2014) Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells. PLoS ONE. 9(4): e96279
(2) Fan, Y., Luo ,Y., Chen, X., Li, Q., Sun, W. (2012) Generation of Human 𝛽-thalassemia induced pluripotent stem cells from amniotic fluid cells using a single excisable lentiviral stem cell cassette. Journal of Reproduction and Development. 58(4): 404-409