Background:
CMU Provost Lori Weingart introduced Dr. Jennifer Doudna as the 2018 recipient of the Dixon Prize in Science. The Dixon Prize in Science is awarded to leaders in science for their accomplishments and recognizes new scientific trends and directions. Dr. Doudna’s lecture will focus on CRISPR systems, a topic of great scientific interest.

Appreciation:
CMU expresses gratitude to Professor Emeritus Guy Berry and the Dixon Prize Committee for selecting Dr. Doudna as the prize recipient. Dr. Erin Mitchell is acknowledged for bringing Dr. Doudna’s candidacy to the table and serving as the faculty host for her visit.

Recognition:
Trustee Dr. Michael McQuaid is recognized for his presence and contributions as a member of the board of trustees.

Event Significance:
The Dixon Prize Lecture is a prominent annual scientific event at CMU that honors leaders in science and showcases important scientific trends. The prize was established by Dr. Joseph Dixon and his wife, Agnes Fisher Dixon, to bring prestige and honor to the universities and the city of Pittsburgh.

Historical Perspective:
CMU has awarded the Dixon Prize since 1970, bringing some of the country’s most forward-thinking scientists to the Pittsburgh campus. The scientific fields recognized have transformed significantly over the past 48 years.

CMU’s Role:
Through the Dixon Prize, CMU has witnessed and participated in this era of scientific transformation, a privilege for its faculty, students, and the entire university community.

Dixon Prize Criteria:
The prize recognizes leaders in diverse scientific areas whose work contributes significantly to their fields. Preference is given to researchers whose work relates to or complements programs at Carnegie Mellon.

Introduction of Jennifer Doudna:
Jennifer Doudna is a renowned scholar and scientist, known for her pioneering work in gene editing and CRISPR technology. She is a professor at the University of California, Berkeley, and a member of prestigious organizations like the Howard Hughes Medical Institute and the Lawrence Berkeley National Lab.

Doudna’s Discovery and CRISPR-Cas9 Technology:
In 2012, Doudna and her colleagues made a groundbreaking discovery, introducing a new method for editing DNA using an RNA-guided protein found in bacteria called CRISPR-Cas9. CRISPR-Cas9 simplified and accelerated the process of editing genomic DNA, opening up possibilities for gene editing applications in humans and other organisms.

Impact of CRISPR-Cas9:
CRISPR-Cas9 technology has revolutionized the field of gene editing, allowing researchers to make precise edits to DNA strands. This has led to advancements in crop and livestock applications, as well as potential treatments for serious chronic diseases like HIV, sickle cell disease, and muscular dystrophy.

Ethical Implications of Gene Editing:
Doudna’s work has also sparked discussions on genetic ethics, as CRISPR-Cas9 could potentially be used for creating “designer babies” or altering human genomes in ways that raise moral and societal concerns. Doudna actively engages in conversations about the ethical boundaries of gene editing and the responsible use of this technology.

Doudna’s Accomplishments and Recognition:
Doudna has received numerous awards and honors for her groundbreaking work, including the Breakthrough Prize in Life Sciences, the Heineken Prize for Biochemistry and Biophysics, and the Japan Prize for Original and Outstanding Achievements in Science and Technology. She was elected to the National Academies of Sciences and the Institute of Medicine, and is a foreign member of the Royal Society.

Doudna’s Approach to Research:
Doudna’s methodology draws from various disciplines, particularly structural biology, to elucidate the mechanisms behind gene regulation and cellular processes. She is known for her ability to make connections between structural discoveries and biological activities, leading to the development of variant proteins with improved functions.

Inspiration from Doudna’s Work:
Doudna’s pioneering work in structural biology highlights the importance of choosing the best tools to address compelling and significant questions, even if it requires venturing into unfamiliar territories. Her journey serves as an inspiration to researchers to pursue their scientific curiosity and make groundbreaking discoveries.

Inspiration from a Book:
Jennifer Doudna’s interest in science was sparked by reading a book that explained how scientists could study and understand complex structures like DNA. This led her to pursue a chemistry major with a focus on biochemistry in college.

The Central Dogma of Biology:
Doudna learned about the central dogma of biology, which states that cells encode genetic information in DNA, copy it into RNA, and then translate RNA into proteins. Initially, she focused on DNA and proteins, considering RNA as a less interesting intermediate.

Discovering the Significance of RNA:
In graduate school, Doudna joined Jack Szostak’s lab, where she became interested in the role of RNA molecules in the origin of life. This marked a shift in her research focus towards understanding the functions of RNA in cells.

Studying Catalytic RNA and MicroRNAs:
Doudna’s early research focused on catalytic RNA molecules and their ability to perform chemical reactions in cells. Later, she studied microRNAs, which are small RNA molecules that control gene expression and play roles in defending against viruses and regulating protein production.

Transition to CRISPR Research:
Doudna’s work on microRNAs led her to CRISPRs, as she sought to understand how RNA molecules control genetic information flow in cells. Her interest in CRISPRs began around 2005 or 2006, when she was exploring new avenues of research.

Discovery of CRISPRs:
Jillian Banfield, a colleague of Jennifer Doudna, discovered unusual DNA sequences called CRISPRs in bacterial genomes. CRISPRs consist of repeating sequences of DNA with unique spacer sequences in between, resembling an adaptive immune system. Spacer sequences matched DNA sequences from viruses, hinting at a potential defense mechanism against viral infection. CRISPRs were found near genes encoding CRISPR-associated (Cas) proteins with similarities to DNA repair and manipulation proteins.

Cas9 as a Programmable DNA Cutter:
Cas9 is a protein within the CRISPR system that acts as a programmable DNA cutter. It can be guided by small pieces of RNA derived from the CRISPR array to find and cut specific DNA sequences. Understanding Cas9’s function allowed for its harnessing in gene editing, a technology that enables precise changes to DNA.

Responsible Progress in Gene Editing:
Doudna emphasizes the need for responsible progress in gene editing, considering its potential risks and benefits. Gene editing should be used in a responsible and ethical manner, ensuring its application aligns with societal values.

CRISPR Adaptive Immune System in Bacteria:
CRISPRs serve as an adaptive immune system in bacteria, protecting them from viral infections. Upon viral infection, the bacterial cell integrates a small piece of viral DNA into its CRISPR array. An RNA copy of the array is generated, and Cas proteins form an effector complex with these RNAs. The complex searches for matching DNA sequences and cuts them, providing immunity against future viral infections.

CRISPR System Diversity:
CRISPR systems in bacteria can be divided into two classes based on their composition. Class 1 systems involve multiple proteins forming effector complexes, while Class 2 systems have a single large protein responsible for RNA-guided defense against viruses.

Collaboration and Unraveling Cas9’s Function:
Emmanuelle Charpentier and Jennifer Doudna’s collaboration led to investigating the function of Cas9, a protein essential for CRISPR function in Class 2 systems.

Cas9’s Mechanism Revealed:
Cas9, together with two separate RNA molecules (CRISPR RNA and tracer RNA), forms a complex that recognizes and cuts DNA. The CRISPR RNA provides targeting information, while the tracer RNA serves as a handle for Cas9 binding.

Single Guide RNA: A Breakthrough in Gene Editing:
Linking the two separate RNAs into a single guide RNA allowed for designing a single molecule with both targeting information and Cas9 binding capability. This discovery led to the realization of CRISPR-Cas9 as a programmable RNA-guided tool for gene editing.

A Transformative Moment:
The understanding of Cas9’s function and the development of the single guide RNA marked a pivotal moment, transitioning the project from fundamental research to a potential gene editing technology.

Publication and Proposal as a Gene Editing Tool:
In 2012, Doudna and Charpentier published their work, proposing CRISPR-Cas9 as an RNA-guided tool for gene editing.

Introduction of Double-Stranded Breaks for DNA Editing:
CRISPR-Cas9 allows precise DNA editing by introducing double-stranded breaks at specific locations in the DNA. These breaks can be repaired by the cell’s natural repair mechanisms, enabling the insertion or deletion of new DNA sequences.

Cas9 Mechanism of DNA Recognition:
Cas9 uses RNA-DNA base pairing to identify the target DNA sequence. The DNA must be unwound to allow for the RNA-DNA hybridization to occur. Cas9 undergoes significant structural changes upon binding to RNA and DNA.

Structural Changes in Cas9:
The protein opens up a channel to accommodate the RNA guide. When bound to a matching DNA sequence, an additional structural change occurs. The cleaver domain in Cas9 rotates into place to cut the DNA.

Precise DNA Cutting by Cas9:
Cas9 only cuts DNA when it’s attached to a matching DNA sequence. This prevents random cutting and ensures precise DNA editing.

Experimental Support for the Model:
Crystallography, electron microscopy, and other experiments support the proposed model of Cas9’s mechanism. Monitoring changes in distance between chemical dyes on the protein’s surface confirms the conformational changes.

Conclusion:
Cas9’s DNA recognition and cutting mechanism involves significant structural changes that allow for precise DNA editing. The protein only cuts DNA when it’s bound to a matching DNA sequence, ensuring targeted and controlled DNA editing.

Cas9 Mechanism and Structure:
Cas9 is a programmable chemical cleaver of DNA that functions in a gene editing context. The protein holds open the DNA strands without external energy and enables RNA-DNA hybridization. A favorable thermodynamic exchange occurs, swapping DNA-DNA hybridization for RNA-DNA, which is more stable. The annealing process between the RNA guide and DNA triggers structural changes leading to DNA cutting.

Challenges in Gene Editing for Curing Genetic Diseases:
Delivery: How to effectively introduce gene editing molecules into the targeted cells. Control of DNA Repair: Understanding and controlling the cellular machinery responsible for editing to achieve precise and consistent results. Ethical Considerations: Addressing the ethical implications of gene editing, particularly for certain applications.

Potential of Gene Editing for Neurological Diseases:
Neurological diseases pose a significant challenge for gene editing due to the difficulty of delivering gene editing complexes into the brain. Ongoing research explores innovative approaches to tackle this challenge and harness gene editing for the treatment of neurological disorders.

Background:
Neurological disorders are often caused or exacerbated by specific DNA sequences in cells. Examples include Parkinson’s, Alzheimer’s, Huntington’s, and ALS. Huntington’s disease is a rare degenerative disorder with no cure. Mutations in a single gene cause Huntington’s, often developing in individuals in their 20s to 40s.

CRISPR-Cas9 Delivery Methods:
Encoding Cas9 protein and guide RNA in DNA: DNA is introduced into cells, copied into RNA, then translated into protein. Using viruses to deliver encoded sequences for Cas9 and guide RNA. Direct delivery of messenger RNA encoding the protein, encapsulated in lipid or other nanoparticles.

Advantages of Non-Genetically Encoded Cas9 Delivery:
Avoids long-term persistence of editing molecules in cells. Reduced risk of unintended DNA cuts and undesired edits.

Brett Stahl’s Experiment:
Used purified Cas9 protein assembled with guide RNA in vitro. Added the protein-RNA complex to cultured cells. Engineered cells with a red protein gene initially turned off. Editing complexes activated the tomato gene, turning cells red. The more protein-RNA complex added, the more cells turned red.

Results:
Demonstrated successful editing of cells using non-genetically encoded Cas9 delivery. Established a dose-dependent relationship between the amount of editor added and the number of cells edited.

Protein Cas9 Complexes with Cell-Penetrating Peptides:
Brett’s idea, based on Carlos Barbas’ work on zinc finger nucleases, involved introducing cell-penetrating protein sequences onto Cas9.

Cas9 Complexes with Different Numbers of Cell-Penetrating Peptides:
Brett generated a series of protein Cas9 complexes with varying numbers of cell-penetrating peptides.

Penetrating Peptides Enable Cell Editing:
Mixing cells with Cas9 complexes lacking penetrating peptides resulted in no editing without artificial disruption of cell membranes. Cas9 complexes with penetrating peptides showed red cells, indicating editing.

Quantification of Editing:
The more penetrating peptides on the Cas9 surface, the greater the editing observed. Gel analysis quantified the extent of DNA changes in cells treated with Cas9 complexes.

Functionality of Modified Proteins:
Modified proteins with penetrating peptides exhibited similar chemical behavior to non-modified proteins.

In Vivo Testing:
Injection of modified Cas9 proteins into a TD tomato reporter mouse resulted in editing of the tomato gene in the mouse’s genome.

Therapeutic Applications:
CRISPR-Cas9 gene editing holds promise for treating neurodegenerative diseases like Huntington’s disease. Researchers have successfully edited about 30% of cells in a given volume using CRISPR-Cas9 in a mouse model of Huntington’s disease. Experiments are underway to determine if CRISPR-Cas9 editing can provide therapeutic benefits to these animals.

Fundamental Biology Observations:
While CRISPR-Cas9 effectively edits different types of neurons, it does not edit astrocytes, a different type of cell in the brain. Despite being in close proximity, astrocytes exhibit a distinct response to CRISPR-Cas9 compared to neurons. Researchers are investigating the underlying mechanisms behind this difference in response to understand fundamental cellular processes.

Responsible Progress in CRISPR-Cas9:
CRISPR-Cas9 gene editing has been applied to a wide range of organisms, including plants, animals, and microorganisms.

CRISPR-Cas9 Technology Revolutionizes Genetic Manipulation:
CRISPR-Cas9 has revolutionized genetic manipulation in plants, animals, and fungi. Researchers can now edit and study organisms at a detailed level. The pace of research has accelerated with CRISPR-Cas9 and other imaging and DNA sequence analysis technologies.

CRISPR-Cas9 in Agriculture:
Researchers have used CRISPR-Cas9 to edit tomato plants to carry a heavier yield. This technology promises precise gene editing in agricultural products to improve traits. It eliminates the need for lengthy breeding processes and minimizes random mutations.

Germline Editing:
Germline editing involves DNA changes in eggs, sperm, or embryos that can be passed on to future generations. CRISPR-Cas9 has made germline editing possible in mice, rats, monkeys, and potentially humans. This has raised ethical questions about “designer babies” and the responsible use of this technology.

National Academies Report on Human Genome Editing:
The National Academies report recommended cautious but not complete cessation of human germline editing. Recent research groups are testing the validity and technical aspects of embryo editing. The technology is rapidly advancing, and its implications and potential use in in vitro fertilization clinics require careful consideration.

Innovative Genomics Institute:
Jennifer Doudna co-founded the Innovative Genomics Institute in Berkeley-UCSF. The institute conducts research, provides resources, and addresses the ethical and societal implications of gene editing.

Outreach and Education:
Jennifer Doudna encourages people to visit their website and blog for more information on CRISPR-Cas9 and gene editing. They are developing a CRISPR kit for teaching purposes, in collaboration with addgene.org, to help students understand how gene editing works.

Collaborators and Funding:
Doudna acknowledges the contributions of her lab members, collaborators, and funding agencies. She particularly highlights Emmanuel Charpentier as a key partner in their work on CRISPR-Cas9.

Kinetics of Cas9 Search and Cleavage:
The search space for Cas9 is vast, given the size of the human genome. Live cell imaging experiments show that Cas9 has rapid kinetics and can access highly compacted parts of the genome. However, the exact time it takes for Cas9 to find a target site in the human genome is challenging to calculate due to unknown factors such as the number of Cas9 molecules in the nucleus. Editing events can be observed within two hours of adding Cas9 to cells, but the number of Cas9 molecules involved in the search and editing process is unclear. Cas9 binding and Cas9 cutting are distinct processes, and most off-target sites do not get cut, despite binding events.

RNA Guide Binding and DNA Search:
Once Cas9 finds its RNA guide, the complex forms a high-affinity interaction that remains stable until degradation. The binding, searching, and cutting of DNA seem to have different kinetics. Cas9 rapidly binds and releases DNA during its search, rather than binding and sliding along DNA. This rapid release action may prevent Cas9 from getting stuck in chromatin and allows for efficient searching.

Implications for the Epigenome:
A question is raised about the implications of CRISPR-Cas9 for the epigenome, but Doudna does not provide a direct answer during this segment of the presentation.

Epigenetics and Cas9:
Epigenetics involves DNA chemical modifications, like methylation, controlling gene expression without DNA sequence alteration. Cas9 can facilitate precise DNA manipulation, enabling targeted epigenetic modifications.

Advantages of Epigenetic Modifications:
Epigenetic modifications may be a safer approach for clinical applications compared to DNA cutting, reducing concerns about DNA recombination and cancer risks.

Cas9 Origins and Prevalence:
Most commonly used Cas proteins for gene editing originate from human pathogens like Staphylococcus and Yersinia. Archaea have CRISPR systems but lack Cas9 proteins of this type. The reasons behind the effectiveness of Cas9 from pathogenic bacteria for gene editing are not yet fully understood.

Detectable Antibodies to Cas9:
Recent research suggests that some people may have detectable antibodies against Cas9 proteins, potentially due to prior exposure to bacteria carrying these proteins. The impact of these antibodies on Cas9 functionality is still unknown.

Future Prospects:
There are numerous unexplored Cas9 proteins in nature that could be tested for gene editing applications. Ongoing bioengineering efforts aim to engineer Cas9 proteins with improved properties. The potential limitations posed by antibodies against Cas9 are unlikely to hinder the progress of this field.