Principles of Site-Specific Recombinase (SSR) Technology

Hey, my name is Frank Buchholz. I'm a group leader here at the Max PL Institute for Molecular Cell Biology and Genetics. And our work is maintained.

We have two aspects of our work. One is concerned about RNA I screening. The other one is concerned about developing site specific recombinase for advanced genome engineering.

My Background is that I'm a biologist, a molecular biologist, and I studied in gutting in biology. I went then on to do a PhD at the European Molecular Biology Laboratories in Heidelberg, and then did my postdoc in San Francisco at UCSF with Mike Bishop working on animal models for leukemia. The Max Puncture Institute here in Dresden tries to combine, let's say, at least two different aspects, which is developmental biologists and cell biologists within the attempt basically to, I think what one could say is that their basic, basic theme is to try to understand how cells form tissues.

That's basically the overall goal of the, of the institute here in Ston. So we work on, On two fields basically. One field concerns doing RNAi screens, trying to identify novel functions of previously uncharacterized genes in biological processes.

The other one deals with genomic engineering to try to develop technologies that allow sophisticated engineering of genomes using site-specific recombinase in specifically, Although Site-specific recombinase were discovered, I would say in the eighties, for instance, the cre recombinase that we work with were, was discovered in the early eighties. It's actually bacterial phage protein. So it originally comes from a bacterial phage, so a, a a virus basically that infects bacteria.

So it's very far away from what is now used at, but it was very rapidly recognized that these enzymes can fulfill very interesting functions that can be used in other organisms as Well. So site-specific Recombinase are proteins that bind to DNA, they bind specifically to a certain sequence of DNA, and when they bind to these target sequences, they're able to cut the DNA and then also rearrange the, the, the sites so that they also basically perform the process of ligation. And again, so you can rearrange DNA sequences with these site specific recombination similar to, you know, scissors and glue that you basically can put together DNA sequences in a, in a, in a way that they were not to put together Before.

So these Site-specific recombinase, as I said, originally discovered in b in bacteria or in bacteria. Phages were then tested to see if they also work in other organisms. And interestingly, they performed their function also in other organisms.

So if you have a site specific recombinase target site in an organism which you have engineered into it, for instance, then these enzymes will fulfill their function. The very prominent use of these site specific recombinase, in particular, the ones that don't need any cofactors, like the flip recombinase or the cre recombinase that we have used as a starting point for the, for the work that we are talking about here today. They work without cofactors.

And if you express them in, in an, in an animal, for instance, or in cells of an animal, then they can find the sequences that they recognize in this organism and then basically cut and paste the right there and, and excise certain sequences. So what has been done or what's where, where this enzyme is, is very successfully been used in so-called conditional mutagenesis of mice, for instance. So let's say you have a gene, which you know is an, is an interesting gene, and you want to know what the function of this gene is.

A very prominent experiment is to do a so-called knockout mouse. For those you have recently seen, this technology has also been giving the Nobel Prize actually this year to do, to basically do a gene deletion in a mouse. Sometimes though, you find that when you knock out a gene in an animal that this is an essential gene and that the mouse will die very early during embryogenesis.

Now this tells you that your gene is very important, but it doesn't really tell you what the function is. So a very prominent way today is to find out, is to do a so-called conditional knockout. So what you do is you flank your gene of interest with target sites for these site-specific recombinase.

And that means that in these mice, the, the gene will be expressed like in wild type mice. But now if you cross these mice with a mouse that expresses the CRE recombinase, for instance, under a tissue specific promoter, then you can delete the gene of interest in only specific cells that you can predefine based on where the recombinase is expressed. And from these experiments, we also know that these recombinations can be very efficient in deleting sequences from whole organisms.

And this was a prerequisite for our idea of trying to develop a recombinase that cuts in the site, which we predefined, which was so far one of the major limitation of further developing the site. Specific recombinase, you had to engineer the recognition target site into the genome of an cell of an organism that you wanted to use. So we thought, can we break this barrier?

Basically using molecular Evolution. Right Now, site specific recombinase are not used for therapy, to my knowledge. As, as I said, that's mainly because you will not have a target site in the human being that will be recognized by any of the recombinase that are available by nature.

So they have to be changed first and able to use them in a therapeutic setting. Now we're only at the very beginning that this may be possible at all. So time will tell if we are really able to develop these enzymes for our, the therapeutic use and time will tell if that's possible or not.