Emmanuelle Charpentier: from tracrRNA to CRISPR-Cas9

Whenever the word CRISPR-Cas9 comes up, the name Emmanuelle Charpentier follows. Prof. Dr. Renneberg traces the career of the Nobel Prize winner.

Translated from the original German version.

The career of Emmanuelle Charpentier

She grew up in a southern suburb of Paris, in rather modest circumstances. Her father was a guard in the nearest park, her mother a head nurse in the administration of a local hospital's psychiatric ward. Emmanuelle once told me a story about her 12-year-old self. One day she was walking with her mother past the world-famous Pasteur Institute. "I'm going to work here, mum... when I grow up," little Emmanuelle shouted confidently.

In fact, as Emmanuelle predicted, she went on to do research on antibiotic resistance in bacteria as a doctoral student at the Pasteur Institute, a field of study that is still a worldwide challenge today. After that experience, her years of teaching and travelling began.

Streptococcus pyogenes: important role for CRISPR

Her first destination was America, where she joined the Rockefeller University in New York, to work with Professor Elaine Tuomanen. When the entire team relocated to the St. Jude Children's Research Hospital in Memphis, Emmanuelle went with them. Later, in warm Alabama, the only thing that bothered her was the mosquito infestation: "apparently the mosquitoes loved sweet French blood..." Emmanuelle said to me when she visited me for the first time in Hong Kong.

Once she returned to New York University, she began working on mouse genes that regulate hair growth. This then shifted to work on rather "unappealing" bacteria that cause skin and throat infections and have the horror moniker of "flesh eater": the Streptococcus pyogenes. These streptococci would later play an important role in the CRISPR story.

Without tracrRNA and CRISPR

After six long years in America, she came back to Europe in 2002, to the lively and noisy Vienna. But soon after setting up her new group, she left Austria again and went to silence and solitude, in the Umea region in northern Sweden. "There I had time to think...", the later-Nobel-Prize winner told me. Emmanuelle had internalised and lived by Louis Pasteur's mottos like "always be prepared for the unexpected!", "happiness favours the prepared mind!". She also believed the motto: "travel educates!" 

Emmanuelle was in the middle of moving to Sweden, when she got an email from Vienna: "Without tracr RNA, no production of crRNA!" She spent a whole night designing experiments, completely obsessed with tracrRNA. After her boss left, the students in Vienna naturally had little desire or time to research tracrRNA in further detail, and Emmanuelle received many rejections as she continued to work on this subject.

tracrRNA chops up long RNA chains into smaller crRNA snippets

Fortunately, graduate masters student Elitza Deltcheva, from Bulgary, came forward. Polish Krzystov Chylinski also joined in. This European team quickly found out that the CRISPR-Cas9 system only needs three components: Cas9 enzyme, crRNA and tracrRNA.

The tracrRNA fragmented long RNA chains into smaller crRNA snippets. These targeted (as if equipped with a GPS) specific locations in the DNA of the attacking viruses. An article was prepared for Nature, and it was published in March 2011, with Elitza Deltcheva heading as the first author. In October 2010, Emmanuelle presented the news at a CRISPR conference in the Netherlands: tracrRNA did indeed play an important role. Charpentier's team knew it first. What she needed now was help from a biochemist like Jennifer Doudna in California.

S. pyogenes has more RNA

Charpentier was the first to understand how the CRISPR/Cas9 type II system works. At the time of her pioneering work at Umeå University in northern Sweden, she was working on the human pathogenic organism Streptococcus pyogenes, trying to understand how its virulence genes might be regulated by small RNAs. There are many such regulatory RNAs in S. pyogenes. Charpentier and her colleagues decided to identify them all. While sifting through the authentic RNA ecosystem, one small RNA caught their interest whose gene was located in the neighbourhood of a CRISPR type-II locus. 

They wondered, since it had to be a Cas gene, what role did it play in CRISPR-mediated immunity? All CRISPR systems known at that time were quite complex. They used many proteins, but only one specific RNA molecule, CRISPR RNA (crRNA). But, it turned out, S. pyogenes had another RNA.

Charpentier recognised the potential for biotechnological applications

Emmanuelle Charpentier intuitively felt that the Streptococcus pyogenes system was different with its extra small RNA. What if this extra RNA actually did one of the tasks performed by proteins in other CRISPR systems? What if this new RNA (which would later be called trans-activating or tracrRNA) interacted with the crRNA to direct the DNA-cutting nuclease to a specific sequence in the genome?

It turned out that Charpentier was not only correct in her intuition, but also that the system she discovered was extraordinarily simple, consisting of only two RNAs, crRNA and tracrRNA, and one protein, Cas9. This meant that it could potentially be transformed for biotechnological applications! It was Emmanuelle Charpentier again who first realised all this.

A controlled DNA cutting machine

In early 2011, Charpentier first met with structural biologist Jennifer Doudna, who worked at the University of California at Berkeley. Doudna, who had studied the structures of different CRISPR/Cas enzymes, was the ideal scientific partner for Charpentier to understand in more detail how the system worked and how it was constructed. Together, Charpentier and Doudna's teams uncovered the sequence of events at the heart of CRISPR-mediated immunity.

The first step is the formation of a partial tracrRNA:crRNA duplex. RNA molecules are usually single-stranded, but their bases can pair like DNA. The double-stranded region of the duplex consists of non-variable tracrRNA and crRNA sequences.

But part of the crRNA remains single-stranded. The sequence of this part is variable and corresponds to the target. This variable part acts as a guide for Cas9, a "microbial GPS" so to speak, to make an exact cut of the DNA double strand, and only where it is needed. The design of the controlled DNA cutting machine was completely logical.