‘On the same wavelength’

Jennifer Doudna credits her laboratory’s success
to someone she took a chance on 20 years ago

What is CRISPR?

It stands for: clustered regularly interspaced short palindromic repeats.

If you had asked Jennifer Doudna a few years ago about the gene-editing tool CRISPR, she would have described the research as “a pretty small effort in my lab, just a few people having fun checking it out.”
 
That was before a lightbulb went off, before Doudna and her postdoctoral fellow realized that the bacterial defense system could be exploited to fix faulty genes or bestow new functions on existing genes in all kinds of cells. The small effort in Doudna’s lab at the University of California, Berkeley, has since exploded into an international one, with laboratories in several continents furiously working on the gene-manipulation possibilities presented by the system. The method has received much attention in the mainstream press, including the New York Times, which described the adoption of CRISPR by researchers as a “scientific frenzy.”
 
Doudna, who is also an investigator with the Howard Hughes Medical Institute, has found herself to be repeatedly sought after as a speaker and has received numerous accolades. Already a member of the National Academy of Sciences and an American Academy of Arts and Sciences fellow, Doudna won the inaugural Mildred Cohn Award in Biological Chemistry from the American Society for Biochemistry and Molecular Biology last year (for work she accomplished prior to the CRISPR craze). This year, she received the Lurie Prize in Biomedical Sciences from the Foundation for the National Institutes of Health. The annual prize recognizes outstanding work by a scientist age 52 or younger; Doudna won recognition for the work on CRISPR.

Doudna Whiteboard
Jennifer Doudna’s laboratory was one of the first to work on the CRISPR/Cas9 gene-editing system.

Partnership

It is the morning after the Lurie Prize banquet, a mild spring day in Washington, D.C., with a chance of rain showers, when Doudna and I sit on an outdoor patio at the Ritz Carlton in Georgetown. Doudna’s husband, Jamie Cate, and her preteen son are upstairs in their hotel room packing to fly back to California while Doudna speaks with me. Friendly and warm, Doudna exudes quiet, deep-rooted confidence. Dressed in crisp jeans and a light gray cardigan, she speaks thoughtfully, occasionally using her hands against the tabletop to make her points in measured sentences. Aware that she has a highly successful research portfolio that covers CRISPR, RNA interference and translational control, I ask how she manages to maintain such a prolific environment in her laboratory of 30 scientists. But Doudna is quick to reveal her secret – her laboratory manager, Kaihong Zhou. “She is the type of person who will do whatever it will take to make the lab successful,” says Doudna. “I owe her a tremendous amount for what we’ve been able to do.”
 
Fresh out of a postdoctoral fellowship with Tom Cech at the University of Colorado, Boulder, in 1994, Doudna was starting as an assistant professor at Yale University. Zhou also was new to New Haven; she had accompanied her husband for his postdoctoral position at Yale. She was searching for a job at the medical school, fully acknowledging that at that time she didn’t know much about biology. Eschewing the human resources department, Zhou went door to door in the medical school asking faculty members if anyone would be willing to hire her. One faculty member said he just had filled his open position for a technician but recalled that a new faculty member was searching for one. He called Doudna to confirm and sent over Zhou’s résumé.
 
Doudna failed to see any common scientific ground in Zhou’s résumé but decided to give her a call anyway. Within 15 minutes of getting the call, Zhou was in Doudna’s office. “She had these big neon-green glasses on and a very bright dress,” remembers Doudna. “She was full of energy. She had no idea what my research was about, but she was eager to learn. She said, ‘If you hire me, I will be in your office tomorrow at 8 o’clock unpacking boxes.’ I thought, ‘Wow, this person is just amazing.’ I gave her a chance.”
 
Zhou showed up the next day even though her paperwork still had to be sorted out in Yale’s human resources department and she couldn’t get paid for another month. Zhou describes herself as restless: “I can’t sit anywhere for more than 20 minutes.”
 
The two women hold each other’s scientific prowess in high regard. “She has great vision and foresight for science,” says Zhou of Doudna; Doudna calls Zhou “a spectacular scientist.” Neither is a procrastinator. If a good idea comes up, the two prefer to set the idea into action immediately. Doudna, describing Zhou and herself as “two peas in a pod,” says, “We confer on all sorts of things, everything from who to hire into the laboratory, how to spend our funds, the kinds of science projects we’re doing and key experiments that need to be done at a particular time.”
 
An ambitious but supportive environment that’s based on teamwork is important to Doudna. “I want the lab to be a place where people feel like they are all batting for the same team.” The sports analogy comes to Doudna easily – her lab has a softball team and makes regular trips to Major League Baseball games (although Doudna is fonder of the social aspects of baseball than the actual sport). “As I’ve chosen people to join my group over the years, I’ve always tried to pick those who would foster that kind of environment,” she says. “For the more senior members of the lab, they need to understand that part of their role in the lab is to provide mentorship to younger students.”
 
And here is where Doudna says Zhou’s presence is important: “She has maintained the kind of environment where students are pushed to do their very best but are also encouraged to seek help when they need it, to understand that they have the support of people in the laboratory when they run into technical challenges or anything else.”
 
In 2001, UC Berkeley offered faculty positions to Doudna and Cate, who was at the Massachusetts Institute of Technology at the time. The offer was tantalizing: Doudna would have a joint appointment to the departments of molecular and cell biology and chemistry. Plus, Berkeley is just a five-minute drive from Lawrence Berkeley Livermore National Laboratory, a place indispensible for a structural biology laboratory like Doudna’s. Furthermore, Doudna’s mother was in Hawaii, so California would bring Doudna closer to her. They accepted.
 
Zhou decided to go with Doudna to the West Coast. Doudna’s research into RNA appealed to her, but Zhou says that their relationship was the key factor. Still, it wasn’t an easy decision for Zhou, because she had her husband’s career to consider. They decided the move was worth the effort. Zhou was the only person from Yale to move with Doudna.
 
Doudna wholeheartedly gives credit to Zhou as an equal partner for making the laboratory successful. And life has taught Doudna that having a true partnership in her personal life also is hugely important. She speaks of Cate: “I didn’t understand when I was younger the importance of having the right partner in life. I only realized it later. I now feel I do have the right partner in life, and I’m very, very, very grateful for that.”
 
She emphasizes that choosing the right life companion is especially important for female scientists. “It’s very important to have a partner who can understand your passion,” she says. “We’re a little bit crazed. We’re driven by what we find exciting in science. I think it’s so important for a partner to understand that level of passion. To make it work between career and family, it’s really critical to have a partner who gets it and is willing to share the burdens.”
Zhou in lab
Kaihong Zhou has been Jennifer Doudna’s lab manager for two decades. Doudna credits her for keeping the lab at the cutting edge of research.

CRISPR: “Small effort” goes big

Doudna always has been interested in RNA. Her research, starting with work she did with her graduate adviser, Jack Szostak at Harvard University, and continued with Cech, has shown over the years that large RNA molecules aren’t a mess of spaghetti-like strings. Instead, they are more like proteins with defined, organized structures. Doudna solved the first crystal structure of a large domain of the protozoan Tetrahymena ribozyme. Her group got the first detailed structure of the P4-P6 RNA fragment of the group I intron, which showed the RNA to be packed tightly into a proteinlike globular fold. In 1998, her laboratory solved the crystal structure of the hepatitis delta virus ribozyme, work that demonstrated how the virus was capable of hijacking its host cell’s machinery to replicate itself.
 
When CRISPR came onto Doudna’s radar in the mid-2000s, her group was working on gene regulation by small RNA molecules in human cells and RNA interference pathways. Doudna became interested in CRISPR, an acronym for clustered regularly interspaced short palindromic repeats, because she realized it was a way for her lab to see how bacteria use small RNA molecules in a pathway perhaps similar to RNA interference. She and her colleagues then could search for any evolutionary relationships between bacterial and mammalian systems in using RNA to control genetic information.
 
Bacteria have three different systems involved in the RNA-mediated destruction of invading bacteriophage genomes by endonucleases. The endonucleases are called CRISPR-associated systems, or Cas. Doudna’s laboratory got involved in studying the type II system when Emmanuelle Charpentier, then at Umeå University in Sweden but now at the Helmholtz Centre for Infection Research in Germany, approached her at a conference. “She wondered if it would be of interest to us to work together to figure out what the function of Cas9 was,” says Doudna. Type I and III CRISPR/Cas systems use a variety of endonucleases; type II is different in that it relies solely on the Cas9 endonuclease.
 
In a 2012 Science paper, the collaborators described how Cas9 attacked bacteriophage DNA. Two pieces of RNA generated from the CRISPR sequences form a structure that Cas9 uses to find the complementary sequence in DNA. Once it finds the complementary sequence, the enzyme introduces double-stranded breaks.
 
That’s what happens inside a bacterium. But one day, while Doudna and her postdoctoral fellow Martin Jinek were discussing some data, they both wondered out loud if they could engineer the two pieces of CRISPR-derived RNA as a single RNA chimera. This chimera could still guide Cas9 to DNA and get the enzyme to cut the DNA as a way of gene manipulation. “We looked at each other and said, ‘If we link these two RNAs together into a single RNA, we will have a very simple two-component system that if we could get it to work in other cells, it would be a very useful tool,” she recalls. “That was the turning point.”
 
The investigators demonstrated a proof of concept in the Science paper using in vitro systems. In the paper, they presciently noted, “Rational design of chimeric RNAs is robust and could, in principle, enable targeting of any DNA sequence of interest with few constraints.”
 
CRISPR isn’t the first gene-editing tool, but its appeal is in its simplicity. All that is technically needed is the Cas9 endonuclease tagged with an RNA strand, which is simple to make in a nucleic-acid synthesizer using synthetic chemistry or enzymes. “Researchers have enthusiastically adopted this system because of the relative ease with which you can manipulate complex genomes compared to other similar technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and other more traditional methods,” explains genomics expert Joel Gottesfeld at The Scripps Research Institute. To date, scientists have used CRISPR to edit genes in almost anything they can get their hands on: human cells, zebrafish, fruit flies, mice, worms and rhesus monkeys.
 
CRISPR gives Doudna a chance to do science with clinical applications. Her hepatitis virus work had a clinical aspect, but the research was far removed from medical applications. Not so with CRISPR. Through an academic collaboration with Hoffmann La Roche, Doudna’s group is looking to see if the CRISPR/Cas9 system can be used to correct known genetic defects in neurological diseases, such as Huntington’s. “We’ve understood the genetic cause for a long time, but up until now, there hasn’t been a good tool for how you might actually fix that mutation,” says Doudna. “To me, that’s very exciting, because it helps us to not only work toward having a direct impact on human health, but I think that when we understand better what the potential limitations are with the current system it will help us as mechanistic biologists to improve the tool further.”
 
CRISPR is also the foundation for the Innovative Genomics Initiative, jointly supported by UC Berkeley and the University of California, San Francisco, and funded by the Li Ka Shing Foundation. The initiative aims to develop genomic analysis to understand disease processes and come up with novel therapeutics. Doudna, who also received a chaired professorship from IGI, is the initiative’s executive director, with UCSF’s Jonathan Weissman serving as a co-director. Although Doudna sees a great future for CRISPR, she says her heart breaks every time she receives an email from someone who has a loved one suffering from a terrible disease or illness asking if CRISPR can help. It’s too early to tell, says Doudna, but the hope is there. Researchers still have to work out the fine-print details, which include figuring out how to target the gene-editing entities into certain cells and not others in a whole organism.
 
Zhou says that if the pace of Doudna’s work was brisk before CRISPR came along, she can only describe it now as hectic. She says the laboratory used to hold an annual potluck, but Doudna’s workload and travel schedule have been so relentless that they had to skip the potluck last year. “She’s super busy,” says Zhou. “She’s become a celebrity!”
Zhou and Doudna
Jennifer Doudna, right, says that she and her lab manager, Kaihong Zhou, are “two peas in a pod.”

Passion and perseverance

The possibility of becoming a scientific celebrity isn’t what drove Doudna to science. Biology infused her childhood, which she describes as “a big adventure.” Her father got his Ph.D. in English literature from the University of Michigan, Ann Arbor. When Doudna was 7 years old, her father completed his thesis and moved his wife and three daughters from Michigan to take up a faculty position at the University of Hawaii. Doudna’s mother, a stay-at-home parent in Ann Arbor who held a master’s degree in education, went back to school to get another master’s degree at Hawaii, this time in Asian history, and began to lecture in the subject at the university.
 
The environmental beauty and excitement of the islands, which included erupting volcanoes, instilled a sense of wonder about the natural world in Doudna. “There were so many fascinating bugs, plants – the natural environment there was so interesting. I was really curious about what makes a plant look the way it does. I always felt very drawn to the underlying mechanisms that work in biology.” At school, Doudna was drawn to mathematics and science. The sense of discovery awed her. She recalls always wanting “to be the first person to know something. That, somehow, inherently was attractive to me.”
 
Her father was a huge influence. “My dad always fostered a sense of curiosity in the house,” says Doudna. Her father loved to read about science, filling the house with books about science geared for nonscientists; when Doudna was in sixth grade, her father presented her with a copy of James Watson’s “The Double Helix.”
 
Her high-school chemistry teacher was also an influence. Doudna, who paid homage to Miss Wong in her Lurie Prize speech, remembers her being “very encouraging and taught kids about the joy of having a question about how does something work and setting up an experiment to test it.” But it was in 11th grade that Doudna discovered what she was meant to do. “The state sponsored a lectureship for people who worked at the cancer center in Honolulu on Oahu to travel around the state and go to high schools and tell the kids what they were doing,” says Doudna. “We had this wonderful woman – I wish I knew her name – who came from the cancer center in Honolulu to my high school. She talked about her work on cancer biology and trying to understand what goes wrong in cells that are cancerous compared to normal cells. That just blew me away. I thought that was so interesting. I absolutely wanted to do that kind of work.”
 
Doudna searched for undergraduate biochemistry programs. “This was in the early 1980s, so there were not that many undergraduate colleges that had biochemistry majors. But Pomona College in Clairmont, California, did, so I ended up going there and starting my work in that direction,” she says. Doudna is the only scientist in the family – one sister is a teacher who is working on federally funded geography projects, and the other sister is an actress.
 
Even though she was inspired to study biochemistry, Doudna remembers having doubts while a sophomore in college taking general chemistry. “It was hard for me, and I was trying to understand why balancing equations was going to be relevant to my future life,” she recalls. At the same time, she was taking a French class and really enjoying it. She approached her French teacher and told her that she probably wasn’t cut out to do science and would be better off majoring in French. Her teacher wouldn’t hear of it. “She said, ‘I can see you’re passionate about it. I know it’s a struggle right now. But you should stick with it. That’s going to be a great career path for you.’ She was right.”
 
The importance of being passionate about the work was reinforced by Szostak during Doudna’s graduate training. Szostak “has a mild, quiet manner to him, but gosh, he could get so excited about science,” says Doudna, adding that his enthusiasm for even the most simple result was infectious.
 
Zhou says Doudna embodies the same upbeat spirit as Szostak. She’s never seen Doudna belittle anyone for experiments gone awry. “Even if something fails, she’ll say, ‘Wow, from this failed experiment, I’ve seen something really great. Let’s try something else from here,’” says Zhou. “She never ever once freaked out because something didn’t work.”
 
Szostak influenced Doudna in another important way. “He would also tell us students in the lab, ‘Follow your passion. Don’t worry about your next career move, because if you follow your passion and do excellent science all of those career decisions will become easy to make, because you’ll know what you want to do and what’s right for you.’ That was so true,” says Doudna. “It’s really guided me in many moments in my career when I’ve had to make a decision.”
 
She respects someone’s enthusiasm for a particular research avenue, because she knows the enthusiasm will help the person persevere. “I think people do their best work when they are very motivated, very excited and passionate about a project,” she says. It’s true for her: Zhou describes Doudna as extremely hardworking, capable of chipping away at work at all hours. “When we are sleeping, Jennifer is working,” says Zhou. “It’s not surprising to get an email from her at 5 a.m.”
 
Doudna is grateful that serendipity also has showed up a number of times in her career. One of those serendipitous moments was meeting Zhou. She emphasizes more than once how similar they are. “We think on the same wavelength,” she says. “Our goals have always been aligned.”
 
Passion and excitement for science is something Zhou says she shares with Doudna. But she adds there is something more fundamental to their relationship – trust and mutual respect. “This is why we’ve lasted for 20 years. We never ever say anything unhappy to each other. It’s amazing. People don’t believe it,” says Zhou. “We’ve been happy together for 20 years!”
Rajendrani MukhopadhyayRajendrani Mukhopadhyay (rmukhopadhyay@asbmb.org) is the senior science writer and blogger for ASBMB. Follow her on Twitter (www.twitter.com/rajmukhop), and read her ASBMB Today blog, Wild Types.

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