Photo: Novo Nordisk Foundation
Disease and treatment

The man with the golden DNA scissors

A tiny bacterium has upended the world as we thought we knew it just a few years ago. It has taken 25 years for researchers to understand what the bacterium was hiding. Virginijus Siksnys, a biochemist in Lithuania, cracked the code. He showed how tiny scissors in the core of the bacterium can be used to cut and paste genes together in a brand new way. This technology has such incredible potential that it may be able to heal sick people and even help save the environment if we learn to use it correctly.

The ability to cut or break down large molecules correctly is an essential process in humans and most other organisms. Sugar has to be cleaved into smaller units before we can use it. Proteins from invading viruses or bacteria need to be cut into smaller fragments so that the immune system can recognize them as being alien in the future. Faulty DNA fragments need to be excised and replaced with the right ones to protect against genetic diseases, including cancer.

The different molecular scissors that carry out these reactions cut small units precisely and rapidly and on a scale that makes all scissors created by humans pale by comparison.

The dream of discovering and controlling nature’s amazing scissors has fascinated the world of research for at least 50 years since the first examples were discovered. Virginijus Siksnys has been involved most of the way. It was therefore fitting that he was the first researcher to understand how to use the most golden of all scissors currently known: the CRISPR-Cas system.

As the uses of this gene technology tool have enormous potential, there are competing claims about its discovery. However, financial rewards and fame have never motivated Siksnys, and his discoveries in this field were very close to not happening.

“After so many years in this field, I had actually become tired of trying to find or engineer the perfect DNA cutting tool that would cut at any desired sequence. I was therefore well on the way to switching my scientific focus when I happened to read an article on the immune system of bacteria. That really made me think – mostly because the bacteria seem similar to humans in their ability to remember the organisms that previously tried to attack them, so they can counterattack effectively the next time they are attacked,” explains Virginijus Siksnys.

The story of the golden CRISPR-Cas scissors is yet another research saga of how seeking to understand nature often results in discovering the most valuable treasures – and often when least expected.

Bacteria’s antiviral defence

Virginijus Siksnys started his golden scissors odyssey in a completely different place. He studied chemistry at Vilnius University and received his PhD from Lomonosov Moscow State University, where he carried out research on enzymes that cut proteins. Returning to the Institute of Biotechnology in Vilnius, he decided to switch the focus and aim at a very fundamental question on how bacteria protect themselves against invasive external virusesknown as bacteriophages.

“Unlike humans, who have many cells, bacteria are unicellular. If a virus gets control of a bacterium, it quickly multiplies and spreads across the whole bacterium population. Bacteria thus have only one try to eliminate an invader, and this requires very effective defence to protect themselves.”

One of the first lines of defence of bacteria are restriction enzymes that can cut DNA into fragments, thereby destroying the DNA of the invading virus. Whether a DNA molecule can be cut depends on the DNA sequence. For example, Escherichia coli has a restriction enzyme, EcoRI, that cuts at the GAATTC DNA sequence. Bacteria protect their own DNA by attaching methyl group tags to the sequence recognized by a restriction enzyme that is unable to cut a methylated target sequence.

“When I started my research career in the 1980s, very few of these enzymes had been identified. However, the enzymes rapidly spread as indispensable tools for DNA manipulation and genetic engineering in bacteria. What interested me and continues to interest me today is understanding how these enzymes achieve their functioning.”

Increasing need for genetic scissors

From 1982 and for two decades, Virginijus Siksnys built up a key centre within this field at the Institute of Biotechnology of Vilnius University. It focused on the structural and molecular mechanisms of restriction enzymes, addressing questions such as how restriction enzymes recognize particular DNA sequences, which common structures are shared by the enzymes and how enzyme structure can be linked to function.

“We hoped that finding answers to these questions would enable us to understand the enzymes’ structures and mechanisms so well that we could engineer them and thereby change their functioning. Ultimately, this could mean that we would be able to design and engineer tailor-made restriction enzymes that would cut precisely where we wanted.”

The need for specific types of genetic scissors grew in the 1990s as gene technology began to gather momentum. The more knowledge obtained about specific genes from the genome of various organisms, the greater researchers desired and needed enzymes to remove genes from one organism and paste them into another.

“Today, we have identified more than 4000 different restriction enzymes that can cut at nearly 300 different sequences, so the diversity is incredibly large. However, although we had improved our understanding of the biochemical mechanisms, and although we could determine the structure by using X-ray crystallography, we could only make limited changes to the enzymes using the technology available at that time.”

Tired of scissors

In those days, the methods for the rational design of enzymes required a lot of protein engineering and were very time-consuming. An enzyme such as EcoRI comprises a chain of 277 amino acids in a specific order. To change the enzyme, researchers had to replace individual amino acids at the protein-DNA interface, test whether the enzyme functioned differently, make another change and test again.

“Although our structural studies implied which amino acids we should change, it was like trying to find a needle in a haystack. In about 2005, I thought I had reached a crossroads since the restriction enzymes seemed to be a dead end.”

Virginijus Siksnys therefore decided to switch tracks with his research, but some unexpected and intriguing research studies occurring around that convinced him to change his mind.

Francisco Mojica, a young researcher in Spain, discovered some mysterious structures in the Haloferax mediterranei bacterium. Mojica found many repeated DNA sequences that each comprised precisely 30 base pairs – separated by precisely 36 base pairs. Mojica became almost obsessed by the structures and spent the next 10 years seeking an explanation. The structures were dubbed clustered regularly interspaced short palindromic repeats or CRISPR.

The CRISPR phenomenon did not create much attention in the next couple of years, but research often accelerates when pioneering scientific discoveries are made, and the CRISPR research suddenly began to take off in 2005. The next significant step convinced Virginijus Siksnys to return to the path he had previously left. The discovery came from a rather unusual source: sauerkraut.

Yogurt and sauerkraut

In France, PhD student Philippe Horvath dedicated his studies to investigating something as exotic as the genetics of lactic acid bacteria used to ferment sauerkraut. Rhodia Food hired Horvath to examine the lactic acid bacteria the company used. Bacteriophages often attacked the bacteria used for producing yogurt, ruining an entire batch.

Horvath had already heard about CRISPR in 2002 and was therefore aware of its possible importance when he discovered the very same genetic system in the lactic acid bacteria he was examining. He decided to look more closely at their genes together with another young PhD student, Rodolphe Barrangou from Danisco USA, Inc. and bacteriophage expert Sylvain Moineau from Quebec, Canada.

Horvath discovered several bacteria that he knew had become resistant to specific bacteriophages. He examined their genome more closely and found that the bacteria had cut out DNA fragments from the bacteriophages and inserted them in their own genome – in precisely the intervals discovered in CRISPR’s repeat DNA sequences.

Virginijus Siksnys explains: “The tiny DNA fragments from the bacteriophages stored by bacteria thus enable the bacteria to recognize the bacteriophages the next time they attack. However, what really startled me was that the cas9 gene in the vicinity of the CRISPR array encoded the Cas9 protein and carried signature sequences seen before in restriction enzymes we had studied previously.”

The bacteria thus apparently place CRISPR – their memory of invading enemies – next to and under something that presumably can cut – the Cas protein. Much more still needed to be understood about CRISPR-Cas and especially its potential, but enzyme expert Siksnys rekindled his interest in bacteria’s immune scissors.

“All my original interest in enzymes arose from the immune system of bacteria. So clearly it was a real eye-opener to suddenly read that bacteria not only had the primitive immune system we had investigated but also have a more advanced immune system.”

A decisive moment

While Virginijus Siksnys set to work studying the possible restriction enzyme in the CRISPR-Cas antiviral defense system, the pieces of the rest of the CRISPR-Cas puzzle slowly began to fall into place.

Researchers from all over the world accounted for individual pieces. John Van de Oost from the Netherlands discovered that the short DNA gene sequences that CRISPR stores to enable it to remember the invading bacteriophages were translated into short RNA molecules (crRNA) that could move around the cell and guide the enzyme cutter to the bacteriophage that needed to be attacked. Emmanuelle Charpentier from Umeå University in Sweden later discovered that crRNA required another RNA guide molecule (tracer RNA) to enable it to find the invading bacteriophages.

In 2011, this knowledge enabled Virginijus Siksnys to become the first person to successfully compile and transfer a complete CRISPR-Cas locus from a bacterium, Streptococcus thermophilus, which is resistant to bacteriophages, into a non-resistant strain of E. coli and showed that this locus enables the E. coli to fight the unwelcome enemy.

“This was a decisive moment for us. We could easily have been missing some components or we might not have been able to transfer the system from one organism to another. But our results clearly showed that we had collected the whole locus and were able to move it around freely. However, we still did not understand how it worked at the molecular level.”

Researchers in the United States, Luciano Marraffini and Erik Sontheimer, had shown that CRISPR-Cas eliminates bacteriophages by cleaving their DNA, and Sylvain Moineau had shown that Cas9 was probably the enzyme scissors that did the cutting. Researchers thus had all the pieces of the puzzle in front of them. They just needed to assemble them.

Hardly believing your own eyes

The race to be the first to assemble the pieces seriously intensified, and deciding who actually crossed the finish line first is still difficult today, but the image that emerged was more magnificent than anyone had expected. After initially cloning the whole CRISPR-Cas system to make it function in another bacterium, Siksnys was ready to focus on his field of expertise: enzymes.

The time had come to determine the mechanism of Cas9, the enzyme bacteria seemed to use to attack the bacteriophages. Siksnys therefore carried out a thorough study with only Cas9-RNA complex and DNA present in a test tube that was the first to confirm that CRISPR-Cas is the enzyme scissors that cuts up the DNA of the bacteriophages. Siksnys’ next discovery was even more surprising and decisive for the breakthrough that CRISPR-Cas subsequently created.

“We strongly suspected that the Cas9 enzyme only cut the DNA fragments if these were identical to the DNA sequences the bacteria had stored from previous attacks. So we thought: What if we change them? Can we make the enzyme cut the sequence we want it to cut?”

Researchers could scarcely believe their eyes. What they had sought for several decades – to reprogramme a restriction enzyme – could now be achieved rapidly. When a 20-base-pair sequence of the crRNA was edited, Cas9 now cut in another location – exactly where the researchers had requested.

Meanwhile, Emmanuelle Charpentier and her United States colleague Jennifer Doudna had achieved exactly the same result, albeit slightly differently, at about the same time. In April 2011, Siksnys submitted his manuscript to Cell. However, the journal rejected it, since they did not find it sufficiently important. He therefore resubmitted it to Proceedings of the National Academy of Sciences of the United States of America, which finally published it on 4 September 2012. Charpentier and Doudna submitted their article to Science 2 months after Siksnys’ submission, but it was published earlier, on 8 June 2012.

The scissors become golden

The two 2012 publications are already landmarks for the life sciences.

“Taken together, these findings pave the way for the development of unique molecular tools for RNA-directed DNA surgery,” wrote Siksnys in his article.

The new results meant that the world now has a gene technology tool that can very accurately edit damaged or incorrect gene sequences and can repair defective genomes by inserting the correct sequences after the DNA is cleaved.

Within 6 months, CRISPR became one of the most frequently searched words on Google. In January 2013, when Boston-based researcher Feng Zhang reported in Science that he had used CRISPR-Cas to edit the human genome, interest on the Internet exploded.

The potential applications for CRISPR-Cas appear to be almost unlimited. Potentially, people with diseases can be healed by repairing the genetic abnormalities, Genetic abnormalities can be introduced into cells to enable researchers to study genetic disorders and thus more easily test new drugs. The new wonder scissors will also make producing drugs, enzymes or other chemicals in cell factories far easier.

CRISPR-Cas has also raised bioethical concerns because it can potentially be used to alter the genome of healthy foetuses. This aspect of CRISPR-Cas technology is still science fiction. Companies are battling for the patents globally, but many researchers are calling for calm and want a moratorium so that the technology cannot begin to be used prematurely.

In the meantime, while the CRISPR-Cas debate rages, one of the chief architects is back in his laboratory in Vilnius – where he started and where he plans to continue for many years to come, that is in basic research.

“In my view, the most important lesson of CRISPR-Cas is that basic research laid its foundations. The key breakthroughs often have the most unpredictable origins. My goal was to understand the antiviral defence mechanisms of bacteria – genome editing came as a second derivative of the basic research. If we had not sharply focused on the fundamental goal, we might never have achieved what we did.”

The 2017 Novozymes Prize was awarded to Emmanuelle Charpentier, Director, Department of Regulation in Infection Biology at the Max Planck Institute for Infection Biology in Berlin, Germany, and Virginijus Siksnys, Professor and Head of the Department of Protein–Nucleic acids Interactions at the Institute of Biotechnology of Vilnius University in Lithuania.

Virginijus Šikšnys
Chief Scientist and Head
Virginijus Šikšnys studied organic chemistry at Vilnius University, receiving his Masters in 1978, then moved to Lomonosov Moscow State University, where he studied enzyme kinetics and received Candidate of Sciences degree (equivalent to PhD) in 1983. From 1982 until 1993 he worked at the Institute of Applied Enzymology in Vilnius. In 1993 he was a visiting scientist in prof. Robert Huber’s laboratory at the Max-Planck-Institut für Biochemie, Martinsried, Germany. Since 1995 Virginijus Šikšnys Šikšnys is the chief scientist and head of the Department of Protein-DNA Interactions at the Vilnius University Institute of Biotechnology, since 2006 – professor at Vilnius University and a member of the Lithuanian Academy of Sciences, since 2007 – chair of the Institute of Biotechnology Council.