Precision genome engineering

What is precision genome engineering? Exactly that! You know what a genome is; ‘engineering’ means that we want to change that DNA, and ‘precision’ means we want to do it in a very specific, targeted way.Sounds challenging, why would you want to do that? Lots of reasons. For example, we might want to make a mutation in a gene in an experimental organism in order to see what the effect was and thus get an insight into the gene’s function. With real precision, we could introduce an exact mutation that corresponds to a human genetic disease allele into an experimental organism. That would allow us to examine the physiological consequences of that mutation in more detail than we could just by looking at people.Are there some more practical, real-world applications? You bet! People are using precision genome engineering with crop plants and food animals to give them improved characteristics. Think about maize that was more drought resistant, or canola that produced more beneficial oil. How about pigs with more muscle mass — call that pork — or dairy cows with no horns to bother their sisters or the farmer. These modifications are already under way.How can you get these changes to be made efficiently and with real specificity? The key is to use proteins that will cut DNA — called nucleases — and to direct them to exactly the place in the genome that you want to modify. The first engineered proteins of this kind were called zinc-finger nucleases, or ZFNs. They have a nuclease attached to zinc finger modules that come from natural DNA binding proteins (mainly transcription factors) and know how to find and bind to very specific DNA sequences. There are natural and synthetic fingers that in the right combination can recognize quite a range of DNA sequences. About four years ago, another type of DNA-binding module was characterized that has a very simple way of recognizing DNA — one module for each base pair. The nucleases made from these are called TALENs (transcription activator-like effector nucleases), and they have stolen quite a bit of territory from ZFNs.So these proteins make a cut in the DNA strand, but what happens then? Once a break is made at a specific site by the nuclease, the cell’s double strand repair machinery hurries to fix it and sometimes makes a mistake. This introduces a mutation right at the break site, and often knocks out the function of a gene. Another type of repair uses a DNA template to copy information across the break. If we put into cells a template that carries sequence changes we want to introduce, they will often get copied in. That’s how you would put in a human disease mutation, for example. Together ZFNs and TALENs have been used successfully to modify the genomes of about 30 different species, including humans.Humans? You’re messing with my genome? No (not yet, but stay vigilant). Lots of genome engineering has been done in cultured human cells, partly to make disease models, partly just to work out the technology. Ultimately we want to use these nucleases for beneficial gene therapy. Right now there are clinical trials going on with ZFNs targeted to the human CCR5 gene. The product of this gene is a protein that the human immunodeficiency virus (HIV) needs in order to infect T cells. It turns out we can get along without this protein — there are natural CCR5 mutants. The therapy is to take T cells from HIV-infected people, treat them in the lab to knock out CCR5, then put them back into the same person. This will prevent the development of AIDS by providing a population of HIV-resistant T cells, and there won’t be a rejection problem because the cells came from the same person who receives them.Sounds amazing, but what can you do besides help AIDS patients? The things that look easiest right now are ones where the nuclease treatment can be done outside the body — ex vivo, as we say. Any condition involving blood cells would be a candidate: thalassemia (hemoglobin deficiencies), severe combined immune deficiencies (SCID), and others. When it comes to delivering the nucleases to intact organs, the challenge becomes greater, but people are working on it. Researchers are also using these nucleases to modify human stem cells in culture. As we learn enough about such cells to have some confidence in putting them into the body, we will also have the tools to correct genetic defects in them. You can imagine manipulating neural stem cells in culture to help reverse neurodegenerative diseases that have a genetic cause.What’s next? There is a lot of work ahead to bring the applications I’ve hinted at to fruition. But beyond the slogging, there is a new nuclease technology, with the catchy name of CRISPR. This system uses a single bacterial protein to do the DNA cutting, and it is guided to its target by an RNA molecule using Watson-Crick base pairing. In that way, it is simpler than ZFNs and TALENs, and it threatens to take over entirely. The CRISPR approach has already been used in a number of systems very effectively, but there are some concerns about whether it will be specific enough for use in people. Keep your eyes open. The future will also be full of surprises.