Learning to edit: the early days of genome engineering
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Almost as soon as the structure of DNA and the genetic code were discovered, scientists started looking for ways to change them, starting with bombarding plants with radiation to cause mutations in the 1960s, before quickly moving on to more sophisticated editing tools - enzymes.
Restriction enzymes, naturally occurring proteins in bacteria that cut DNA, were discovered in 1968 by Werner Arber, a Swiss microbiologist and geneticist who won the Nobel prize for his work in 1978. The budding field of genetic engineering quickly adopted restriction enzymes to create hybrid DNA and modified organisms ranging from bacteria that produce synthetic insulin to longer-lasting tomatoes and GM salmon or even pets. For more on that, take a listen to our previous podcast story GMO? OMG! The history of genetic modification in episode 23 of our first season.
While restriction enzymes were the workhorses on which the molecular biology revolution was built, they have their limits when it comes to changing the genome, because they can only cut DNA into fragments at or near a specific short sequence.
For example, the enzyme EcoR1, isolated from E coli bacteria, cuts DNA wherever it finds the sequence GAATTC,
leaving a short overhang that will match up and anneal with any other piece of DNA that’s been cut with the same enzyme (or another enzyme that leaves the same overhanging ends).
Although there are a wide range of enzymes available with different recognition sequences - though, based on my experience building bits of DNA in the lab, never quite the one you really need - restriction enzymes are still somewhat restricted in cutting and pasting bits of DNA together, rather than making very targeted changes anywhere of your choosing in the genome.
For that, and to fulfil the promise of using genetic editing to prevent or cure genetic diseases, we need more sophisticated tools. The big challenge here lies in designing tools that can find the correct sequence and precisely edit it. It’s like opening a Word document containing a million pages, hitting ctrl+f and finding and fixing a sneaky typo hidden somewhere in the middle.
Luckily, if you can find the sequence you want to fix and make a precise cut in the DNA, the cell will do the rest of the hard work of gene editing for you.
When DNA is cut, the cell will repair it in one of two ways. The first is a process called non-homologous end joining, which introduces mutations at the site where the DNA was cut. Alternatively, if there is another piece of DNA that matches up to the sequence you’ve cut and just happens to be floating around, it will incorporate that piece of DNA to fix the strand, known as homologous recombination. And it’s here that there’s a prime opportunity to fix a faulty gene, or introduce any other alterations or changes.
That’s why precise, programmable genetic scissors that accurately cut DNA at a defined location in the genetic code are the key to successful gene editing.
The first person to recognise this was Srinivasan Chandrasegaran, a scientist from John Hopkins University in Baltimore, Maryland, and in 1996, he found a way to create restriction enzymes that could cut DNA at any sequence you wished - not just at specific recognition sites.
Chandrasegaran created clever synthetic hybrid enzymes combining the DNA-cutting domain from a restriction enzyme with special molecules that bind to DNA, known as zinc fingers.
Each finger binds to 3 ‘letters’, or bases of DNA, depending on its molecular makeup. This means that it’s possible to design ‘hands’ of zinc fingers that will bind to any specific sequence of DNA, effectively making a completely programmable restriction enzyme that snips exactly where you want it.
Although zinc finger nucleases opened the door to targeted gene editing, they are complicated to make and time-consuming to adapt to different target sequences. Engineering them can take months or even years.
Attempting to speed things up, Professor Daniel Voytas, a plant biologist and genetic engineer from the University of Minnesota, turned to plant pathogens and a bacteria called Xanthomonas. The bacteria inject molecules called TAL effectors into plant cells, which then make their way into the nucleus and bind to the plant DNA, sticking to specific sequences and activating gene expression patterns that allow the bacteria to colonise their host.
Understandably, plants are not best pleased by this invasion, and they adapt by selecting for mutations in the TAL effector target sequences. Undeterred, the bugs fight back, switching up their TAL effectors to cope with new mutations in the plant genome. So, Voytas figured, why not harness these adaptable, flexible DNA-binding proteins to create a new genome engineering tool?
In 2010, he fused TAL binding domains with the same cleavage domain used in zinc-finger nucleases to create a new genetic editing technology called TALENS. However, unlike zinc fingers which bind to three DNA letters, each TAL domain only binds to one, making them less complicated and more adaptable than zinc fingers and speeding up the process of adapting the nucleases to new sequences from months to 1-2 weeks.
For a brief moment in the late noughties, zinc fingers and TALENS enjoyed their time in the genetic engineering spotlight. But they were still tricky techniques to get working, so only the most dedicated labs gave them a go. Then in 2011 came the discovery that would change everything: CRISPR
