Doctor Jennifer Doudna is a Professor of Chemistry and Biology and the Li Ka Shing Chancellor’s Chair at the University of California, Berkeley. She is an investigator with the Howard Hughes Medical Institute, a senior investigator at Gladstone Institutes, and founded the  Innovative Genomics Institute, to develop genomic analysis to understand diseases and come up with novel therapeutics using genetic editing.⁵ Among her many honors, Doudna is a member of the National Academy of Sciences, a Foreign Member of the Royal Society, and has received various prices including the Breakthrough Prize in Life Sciences (2015), the Gruber Prize in Genetics (2015), the Kavli Prize (2018), the Wolf Prize in Medicine (2020), and most importantly, the 2020 nobel prize in Chemistry—an honor she shared with French scientist Emmanuelle Charpentier for their research into genome-engineering technology. Doudna has also been recognized as one of TIME’s 100 Most Influential People in 2015.¹ She is the author of the book “A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution”⁴ and currently lives in Berkeley, California with her husband Jamie Cate and their son.

Background

Jennifer Anne Doudna was born February 19, 1964 in Washington. D.C.² to Dorothy and Martin Doudna. When she was seven, her father, mother, and two sisters moved from Ann Arbor, Michigan, to Hawaii so her father, who had a Ph.D. in English Literature, could accept a teaching position at the University of Hawaii in Hilo; her mother was a history lecturer. It was in Hawaii that Doudna fell in love with science: she would take long hikes in the forest, look for shells on the beach, and identify various plants on her walks. “In high school I realized I loved math and loved chemistry,” she recalls.³ Her parents, both academics in the humanities, encouraged her interests and provided her with science books, museum visits, and connections to their fellow faculty at the University of Hawaii.⁶ In her junior year of high school, she heard a lecture from a young female scientist on cancerous cells, and was struck by the idea that she could be both feminine and a brilliant scientist. She spent a summer working in a biology lab under UH Hilo professor Don Hemmes, who she credits with inspiring her curiosity about the living world.⁴ 

Doudna graduated from Hilo High School in 1981 and attended Pomona College in Claremont, California as an undergraduate student where she was further inspired by her various teachers and mentors—notably her undergraduate advisor Sharon Panasenko, whose lab she worked in.³ She almost considered switching her major to French as a sophomore, but ultimately, her French teacher pushed her to stick with science.⁵ Doudna earned her BA in Biochemistry in 1985 and went to Harvard Medical School for her doctorate. She earned her Ph.D. in the laboratory of Jack Szostak, where she developed an interest in RNA, which would later provide the impetus for her research. With Szostak, the two wrote a paper on the reengineering of an RNA self-splicing intron into a ribozyme by studying the exact stereochemical course of an RNA-catalyzed reaction.⁶ In 1988, Doudna married fellow Harvard graduate student Tom Griffin, but they divorced a few years later due to conflicting interests. Doudna earned her Ph.D. in Biochemistry and Molecular Pharmacology in 1989 with her dissertation, which demonstrated that RNA does not just carry genetic information but also helps catalyze the process.⁷ From 1989-1991, she took on a research fellowship at the Massachusetts General Hospital under Szostak. 

In 1991, Doudna went to the University of Colorado Laboratory as a Lucille P. Markey postdoctoral fellow⁸ under Tom Cech, who had just won a Nobel Prize for the discovery of ribozymes. Doudna worked on crystallizing a ribozyme so that, with X-ray diffraction, its 3D shape could be revealed for the first time. Cech says that “when she hits a roadblock—guaranteed in our field—she is adept at finding a path to success.”³ Doudna eventually published a paper showing the shape of a ribozyme with Cech. Her group was the first to detail the structure of the P4-P6 RNA fragment of the group I intron, which proved that RNA was packed into a tight, globular structure.⁵

At the University of Colorado, Doudna met her husband Jamie Cate, who was then a graduate student, and in 1994, when Doudna left to become an assistant professor at Yale University in New Havens, Cate went with her.¹⁰ In 1994, Doudna left Colorado to become an assistant professor at Yale University in New Havens. It was there she met Kaihong Zhou, who Doudna credits wholeheartedly with the success of her lab.⁵ Doudna recalls that “she had no idea what my research was about, but she was eager to learn.”⁵ At Yale, Doudna launched her research group—the research group with which she would eventually make groundbreaking discoveries in gene editing.⁹ In 1998, her laboratory solved the crystal structure of the hepatitis delta virus ribozyme, which demonstrated how the virus could hijack host cell machinery in order to replicate itself.⁵ Two years later, at the turn of the century, Doudna both became a Henry Ford II Professor of Molecular Biophysics and Biochemistry and married Jamie Cate in Hawaii.¹⁰

In 2001, UC Berkeley offered faculty positions to both Doudna and Cate, who was at MIT at the time: the two accepted. At UC Berkeley, Doudna had a joint appointment in the departments of molecular and cell biology and chemistry; Berkeley was further just a five-minute drive from the Lawrence Berkeley Livermore National Laboratory, which was indispensable for a structural biology lab like Doudna’s. Zhou followed Doudna to the West Coast due to their strong relationship—the only person from Yale to move with Doudna.⁵ At UC Berkeley, Doudna pursued her RNA research, and it was while doing so that she grabbed a fateful cup of coffee with fellow Berkeley microbiologist Jill Banfield in 2005, who wanted Doudna’s help in understanding the repetitive CRISPR sequences she had spotted in bacterial genomes she was studying. Doudna was swept up into figuring out how CRISPR worked because she believed it could provide clues into how RNA in human cells regulated genes and pathways, the topic she and her group were then investigating.¹⁰ The idea that bacteria could have a humoral defense in its immune system and record previous diseases went against what scientists believed at the time, which was that bacteria only had a rudimentary immune system. A few years later, in March 2011, Doudna met French microbiologist Emmanuelle Charpentier, who was also studying a CRISPR system in the S. thermophilus bacteria; specifically, the active protein, Cas9.

The two began collaborating internationally on their research into the Cas9 protein. Together, Martin Jink, a postdoctoral student in Doudna’s lab and Krzysztof Chylinksi, a postdoctoral student in Charpentier’s lab, crystallized the Cas9 protein to model its 3D structure and to understand how it worked.³ After months of research, the group published their research on Cas9 in Science Magazine and caught the attention of the international scientific community. Ultimately, in October 2020, Doudna and Charpentier won the Nobel Prize in Chemistry for their development of gene editing. When Doudna won the prize, she said: “I hope that this prize and this recognition changes that at least a little bit, and that it’s encouraging to other women who are in science, or even in other fields, to realise that, you know, their work can be honoured and that their work can have a real impact.”¹⁵

Content and Contributions

Crystal structure of a hepatitis delta virus ribozyme¹⁴

As of the publishing of this paper, the self-cleaving ribozyme of the hepatitis delta virus (HDV) was the only catalytic RNA known to be required for the viability of a human pathogen. HDV is an RNA satellite virus of Hepatitis B. It has a circular, single-stranded RNA genome of approximately 1,700 nucleotides that is replicated by RNA polymerase II in the host. The HDV ribozyme is the fastest known self-cleaving RNA, and is incredibly stable compared to other ribozymes. Ribozymes themselves are catalytically active RNA. Its first-order rate constant is about 52 reactions per minute at 37 °C, which in vitro, is more than 1 cleavage per second. Remarkably, it does not require divalent cations around it such as Ca²⁺ or Mg²⁺ for activity, unlike other ribozymes.

Doudna crystallized a 72 nucleotide, self-cleaved form of the HDV ribozyme by engineering the RNA to bind to a small, basic protein without affecting ribozyme activity. The P4 stem of the HDV ribozyme was engineered to contain a high-affinity binding site for the RNA-binding domain of a spliceosomal protein. The P4 stem is noted to be easy to edit without changing cleavage or catalysis of HDV, which is why it was selected. The structure of the complex was then determined using multi wavelength diffraction. Overall, the HDV ribozyme comprises of five segments nested in a knot. No tightly bound metal ions are found in the HDV ribozyme; the structure is instead stabilized by base-pairing and backbone interactions. The folded structure is incredibly convoluted, but it contributes to the stability of the ribozyme, since disruption of the small pseudoknots in the structure reduces ribozyme activity. The structure that Doudna found was overall an excellent starting point for her research into the incredibly active, catalytic RNA.

Ribozyme Structure and Mechanisms¹³

One of Doudna’s first big research projects was at Yale, a continuation of the research she started at the University of Colorado on the structure of ribozymes. When she published the paper, seven naturally occurring classes of catalytic RNA had been identified, some of which she herself had identified and published on like the hepatitis delta virus as detailed above. The ribozymes were all small RNAs of 50-150 nucleotides that performed site-specific self-cleavage, using base pairing to align with a specific cleavage site. People were originally surprised at the notion that RNA was catalytic (something Doudna again published on at Harvard) since RNA lacks the diversity of enzymes. However, RNA enzymes are capable of catalyzing phosphoryl transfer reactions by 10⁵ to 10¹¹ fold over, and can accelerate a variety of reactions as well. How? 

Normally, proteins like RNase A catalyze fractions via acid-base catalysis, where a histidine group (amino acid group) acts as a base and a second histidine group acts as an acid, donating a proton. The proteins are able to modulate the pKas (a topic from CHEM 131) of the histidines and thus RNAase A accelerates the reaction. RNA does not have the same function groups as other proteins and thus cannot use the acid-base catalysis mechanism to accelerate reactions. Instead, ribozymes can carry out metal-ion assisted catalysis, similar to the actions of other enzymes that catalyze phosphate chemistry. This is because metal ions like Mg²⁺ are essential for RNA folding, so RNA already has the binding sites for divalent ions. Metal ions could activate a hydroxyl via deprotonation, or stabilize a transition state by donating a positive ion. For example, experimentation allowed for the detection of magnesium binding sites in the catalytic cores of several different ribozymes.

CRISPR Cas-9 Structure and Mechanisms¹¹

The CRISPR (clustered regularly interspaced short palindromic repeats) Cas-9 protein is a natural bacterial defense mechanism against infection that can be repurposed as an RNA-guided genome editing tool. It holds promise for treating many genetic disorders, including forms of cancer and neurodegeneration, as well as viral infections or cardiovascular diseases. How does it work? Following exposure to an invader, the Cas9 protein integrates short fragments of foreign DNA into the CRISPR repeat-spacer array to provide a genetic record of prior infection and make it easier to defend against future invaders. Subsequent transcription of the CRISPR array yields CRISPR RNA’s (crRNAs). At the 5’ end, the crRNA has a spacer, and the 3’ end has a piece of the CRISPR repeat sequence. Together, they trigger the destruction of invading DNA by Cas nucleases upon a second invasion.

Cas9 essentially cuts a piece of DNA out of the genome: this can be used to rid the human genome of unwanted sequences that cause diseases. tracrRNA, or trans-activating crRNA, pairs with the repeat sequence of crRNA to make a dual-RNA hybrid structure that directs Cas9 to cleave any DNA complementary to its target sequence. By changing the guide RNA sequence in the crRNA, the CRISPR-Cas9 system can be programmed to target any DNA sequence of interest. The system creates a site-specific, blunt, double-stranded break, removes an unwanted genome sequence, and then repairs the site. The biggest problem is that the site repair can result in small, random nucleotide insertions or deletions. Cas9 binds to the DNA via a PAM sequence that is adjacent to the target site; this is crucial for the system to delete only specific sequences of the genome. Single PAM mutations can disable Cas9 activity entirely. Each target site for editing has a corresponding PAM site that the Cas9 system binds to before beginning DNA cleavage. Cas9 uses the RuvC nuclease domain and the HNH nuclease domain to cleave one strand of the target double stranded DNA, make the change, and then bind the site shut. 

The mechanism for genetic editing is overall incredibly detailed and complicated; furthermore, Cas9 is not the only bacterial protein that has been discovered: Cas12 and Cas13 are both cousins of the protein that Doudna has dedicated her research to.¹² 

Bibliography

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