Something remarkable has happened on the seventh floor of UC Berkeley’s Stanley Hall, where Professor Jennifer Doudna’s lab resides.
Two years ago, Doudna (rhymes with proud-na) and her colleagues published their discovery of a versatile new way to make precise cuts in DNA, the chain of nucleic acids that carries the genetic code of living things. The method relies on a tiny protein that evolved in the immune systems of Streptococcus bacteria. Working like a pair of scissor blades, this clamshell-shaped protein protects bacteria by tracking down and slashing the DNA of viruses that prey on the bacteria. Now this Lilliputian virus-killing machine, which goes by the ungainly acronym CRISPR/Cas9, has been repurposed by Doudna and her team and it is transforming molecular biology research throughout the world.
“I have never seen a technology take off so quickly. It has set the community on fire.” - Dr. Bruce Conklin
It is difficult to overstate the importance of Cas9 gene-slicing techniques. They not only speed up tedious laboratory processes, but also open up new possibilities for medical treatments—reviving hopes for gene therapies and perhaps a new generation of antibiotics. Already, the technology has been adopted as a laboratory standard.
“It’s like a dream come true for me as a biologist,” says Craig Mello, the University of Massachusetts researcher who shared the Nobel Prize in Medicine in 2006. “This overcomes a serious bottleneck. It allows us to alter genes effortlessly, almost at will.”
Doudna came to UC Berkeley from Yale in 2002 with a reputation for working side-by-side with Nobel laureates and having a knack for building alliances with other creative thinkers. She was also known for her brilliance at teasing out the purpose of biomolecules and for an uncanny ability to glean the shapes of the virtually invisible: the remarkable molecular machinery that spins within living cells. She was lured to Berkeley not only by what she calls its “interesting pioneering spirit,” but also by its proximity to the U.S. Department of Energy’s Lawrence Berkeley National Laboratory. This is where the giant Advanced Light Source overlooking the Cal campus produces the kind of high-intensity X-ray beams needed to probe the complex structure of proteins and other molecules. [Disclosure: The author is a former employee of the Berkeley Lab.]
Breaking up segments of DNA is fundamental to research in molecular biology. The basic idea is that if you can cut, disable, or change a segment of DNA from the gene of a cell, you can figure out the function of that segment by observing what happens to the newly made mutants. It has been painstaking work—slow, inefficient, and expensive—carried out with the lab equivalent of a hammer and saw. Doudna and her collaborators have provided a scalpel.
“This is a genome-editing tool,” she says. “We’ve been able to read and write DNA for a long time. We have machines to sequence it (read); and to synthesize it (write). What we haven’t been able to do is to rewrite it—to edit it. And now we have a tool that lets you do something about that. It enables many kinds of experiments that were not possible in the past. That is incredibly exciting.”
In the laboratory, efficient genome editing means time and money saved. It can take a year or more to develop an engineered strain of laboratory mouse that mimics a human genetic disease. With Cas9, that can be accomplished in six weeks, giving scientists a quick way to test potential new therapies. The technology meshes well with stem cell science, allowing the rapid probing of signals that prompt these master cells to become different kinds of tissue and organs.
In the clinic, Cas9 technology is raising new hopes for gene therapy, a once-promising approach to swap bad genes for good ones, which ran into safety problems—among them: a gene inserted in the wrong spot can cause tumors. At the Gladstone Institutes in San Francisco, UCSF professor Dr. Bruce Conklin says the capability to accurately edit genomes has rekindled enthusiasm. “I have never,” he says of Cas9, “seen a technology take off so quickly. It has set the community on fire.”
The first genome editing therapies will focus on “monogenic” diseases, which are caused by a single defective gene and hence are potentially the easiest to correct by rewriting. There are literally hundreds of these diseases; among the better known are Huntington’s disease, sickle-cell anemia, and cystic fibrosis. Conklin cautions, however, that Cas9 accuracy, fine for the lab, is still not good enough for the clinic. Much of the research in genome-editing therapeutics is now focused on tweaking Cas9 to improve its accuracy, or on developing other cutting technologies, all to reduce potentially deadly “off-target effects.”
Meanwhile, researchers at MIT have used Cas9 to target bacterial genes that confer resistance to antibiotics. Such work is of critical importance because decades of medical progress is threatened by the rise of antibiotic-resistant strains of bacteria. Experiments on these bacteria show that Cas9 can be directed to knock out the production of enzymes with which the microbes blunt the effect of antibiotics.
Applications for Cas9 extend beyond the medical field. Jamie Cate, a Berkeley professor who also works at the nearby Energy Biosciences Institute, is using Cas9 to alter yeast used in the production of biofuels. “You want to work with hardened, industrial-capable yeast able to survive harsh conditions,” Cate explains. With Cas9, he recently produced a strain that boosted fermentation of cellulose more than tenfold.
Cate is a fan of Doudna for other reasons, as well. He first met her when he was a grad student at the University of Colorado, where she was a postdoc. They later married when she was teaching at Yale. Now they are both Cal professors and have an 11-year-old son, Andrew, who likes computers and math; he attends middle school in Berkeley.
The team understood that this customized “guide RNA” might be directed to cut precisely any piece of DNA, from any organism. That was the moment when Doudna felt the hair stand up on the back of her neck.
It was as a young girl living in Hawai’i that Doudna first became acquainted with the joy of scientific discovery. She grew up on the Big Island, where her father taught American literature at the University of Hawai’i at Hilo; her mother was a history lecturer. Doudna took long hikes in the tropical forests, spotting plants and animals, and prowled the beaches for shells. Don Hemmes, a family friend and biologist at the university, invited her to his lab for a summer of studying worms and mushrooms. Another summer, she learned how to use an electron microscope, an instrument that later became indispensable for her explorations of molecular structures.
“In high school I realized I loved math and loved chemistry,” she recalls. Junior year, she attended a program at a Honolulu cancer center and heard a lecture given by a young woman with long blonde hair. Doudna was mesmerized by the topic—why cells become cancerous—but was also struck “that this feminine person was clearly an incredible scientist. It was an important moment.”
At Pomona College in Southern California, Doudna was further inspired by her teachers—notably Sharon Panasenko, her undergraduate advisor, in whose lab she worked. “Mentors are critical,” says Doudna. “And fortunately for me, I’ve worked with absolutely outstanding scientists at every stage of my career.”
At Harvard she earned her Ph.D. in the laboratory of Jack Szostak, who 20 years later would share the 2009 Nobel Prize in Medicine for his insights on the gene-tending enzyme, telomerase. In Szostak’s lab, Doudna developed a passion for the mysteries surrounding RNA. Scientists then were beginning to realize that this peculiar cousin of DNA held a much greater role inside cells than was previously thought.
That work took her to the University of Colorado laboratory of Tom Cech, who had just won the Nobel Prize for his discovery of ribozymes, RNA molecules that can twist into shapes that catalyze chemical reactions, just as proteins do. Doudna took on the daunting task of crystallizing a ribozyme so that, with X-ray diffraction, its 3D shape could be revealed for the first time. “She’s very imaginative and can see possibilities that others don’t,” says Cech. “She has an uncanny knack for picking the best experiments to answer a question. When she hits a roadblock—guaranteed in our field—she is adept at finding a path to success.”
Doudna left Colorado in 1994 to take a professorship at Yale. She kept up her collaboration with Cech, and they later published a milestone paper showing the shape of the ribozyme. Doudna continued to plow new ground at Yale with her own studies of RNA structures, and carried it forward to Berkeley. “Almost from the start, Jennifer has been a star in the field of structural biology,” says Yale RNA expert Joan Steitz. “Whatever she touches, she gets something golden out of it.”
At Cal, Doudna has pursued what she has called “the secret life of RNA.” If DNA is the monarch of biological molecules, RNAs would seem to be its court attendants. Their most well known function occurs when the double helix of DNA is unzipped, and copies of its exposed genetic code are imprinted on a strip of RNA. This “messenger” RNA is then shuffled off to a cell’s protein-making machinery, which uses it as a template to snap together amino acid chains.
However, new research shows that RNAs are much more than just obedient courtiers—in some ways, they rule the realm. A variety of RNAs act as switches that turn genes on or off. Much of the ribosome, the factory that makes proteins from RNA instructions, is constructed of RNA itself. An entire class of “small, interfering RNAs” can jam genetic machinery, and appear to serve as a shield against foreign microbes by smothering and “silencing” the invaders’ genes.
It was while exploring these RNA defense mechanisms in 2005 that Doudna had a fateful cup of coffee at the FSM Café with Jill Banfield, the Berkeley geo-micro-biologist known for sequencing the genes of microbial life found in harsh environments, including the grossly polluted, highly acidic Iron Mountain Mine near Redding. “Jill told me there might be some interesting biology for me in the wild bacteria she works with,” Doudna recalls. “They were coming across a lot of CRISPR sequences.”
CRISPR stands for “clustered regularly interspaced short palindromic repeats,” an insider’s term for its signature pattern of DNA. CRISPRs were first identified in E. coli bacteria by Japanese researchers in 1987, but they weren’t named until 2002 and their purpose was still unknown. Today CRISPRs are thought to lie in half of all bacterial species and in 90 percent of archaea, a vast separate kingdom of single-celled microbes. CRISPRs have a distinctive presence on the genome: small packets of DNA lined up like boxcars, all of similar size.
With the advent of genome sequencing technology, interest in CRISPRs grew. DNA-reading data showed that those boxcars carried gene fragments from phages, tiny viruses that for eons have been locked in combat with both bacteria and archaea. Some scientists theorized that CRISPRs were an immune defense used to identify and kill invading viruses. The theory was confirmed in 2007, by a team of scientists from yogurt-culture developer Danisco. Now the question was, how does it work?
What piqued Banfield and Doudna’s interest was the prospect that CRISPRs might use some form of RNA interference—a mechanism under study in Doudna’s lab—to kill viral genes. Evidence started pointing to a different process, and the mystery only deepened. Interest focused on a variety of structures called CRISPR-associated (Cas) proteins. To find out more about them, Doudna assembled a team of young researchers.
In 2011, at a scientific conference in Puerto Rico, Doudna met Emmanuelle Charpentier, a French microbiologist then working in Sweden. “She and I really hit it off,” recalls Doudna. Charpentier described a CRISPR system she was studying from a pathogenic strain of strep bacteria, S. thermophilus. Of particular interest was its active protein, Cas9, which seemed particularly efficient at slicing up viral invaders.
An international collaboration blossomed. The goal was to find out what Cas9 looked like and how it functioned. Doudna put one of her young postdocs, Martin Jinek, in charge of a research team. Though on different continents, Jinek’s team worked closely with that of Krzysztof Chylinski, a postdoc from Charpentier’s lab. Together they purified and crystallized the protein and used X-ray beams and electron microscopy to model its 3D structure, which was only five times the width of DNA itself. Cas9 emerged as a sleek protein that carried within it a short segment of RNA that acted like a sensor to guide the protein to a matching sequence of DNA inside an invading virus. Once attached, Cas9 enzymes would break both strands of the target DNA. “That was the first really exciting moment,” Doudna says of the discovery.
“We’ve been able to read and write DNA for a long time. We have machines to sequence it (read); and to synthesize it (write). What we haven’t been able to do is to rewrite it—to edit it. And now we have a tool that lets you do something about that.”
Then Jinek found that he could easily reprogram the Cas9 protein. All that was needed, he discovered, was a short segment of RNA code matched to the corresponding code of the target site, and Cas9 could make the cuts. The team immediately understood that this customized “guide RNA” might be directed to cut precisely any piece of DNA, from any organism. That was the moment, Doudna told NPR’s Joe Palca, when she felt the hair stand up on the back of her neck.
Today Doudna and her team are focused on refining their knowledge of the CRISPR/Cas9 system. “We are really keen to understand how this gets into the DNA, pries open the double helix, and then holds on long enough to let a cell repair the breaks it makes.” They also wonder what changes might improve it. Doudna says she’s “a firm believer that if you understand the way something in nature works, you can begin to use it for different applications; and sometimes you can make it work better.”
In November, Doudna and Charpentier were awarded a $3 million Breakthrough Prize for their work on Cas9. She admits her head is filled with visions of these tiny proteins, her imagination sifting through different shapes, different solutions to the puzzle of their structures. “It’s on my mind a lot. I dream about it. I wake up thinking about it.” For a structural biochemist, it is a puzzle that fascinates, and some of her most productive thinking occurs in those quiet spells when she goes for a run or hikes the Berkeley hills.
“Those insightful moments don’t come along so often,” says Doudna, “which is why we cherish them as scientists.”
Sabin Russell is a freelance science writer. He covered medical science and health policy for the San Francisco Chronicle and was a Knight Science Journalism Fellow at MIT.