Welcome and Introduction:
John Mazziotta, Vice Chancellor of Health Sciences, welcomed everyone to the 56th annual lectureship of the David Geffen School of Medicine. Dr. Jennifer Doudna, a renowned scientist and Howard Hughes Medical Investigator, was introduced as the lectureship award recipient. Dr. Doudna is a Professor of Chemistry and Molecular and Cellular Biology at UC Berkeley and holds the Li Ka Shing Chancellor’s Chair in Biomedical Sciences.

Importance of Scientific Exchange:
The annual lectureship emphasizes the significance of scientific exchange and breakthrough discoveries in biomedical science. The essence of a great university lies in discovery, new ideas, and knowledge that benefit society.

Introduction of Dr. Jennifer Doudna:
Doug Black, Professor of Microbiology, Immunology, and Molecular Genetics, was invited to introduce Dr. Doudna instead of Interim Dean Kelsey Martin. Dr. Black described Dr. Doudna as one of the most admired and influential scientists in RNA biology.

Dr. Jennifer Doudna’s Accomplishments:
Dr. Black mentioned Dr. Doudna’s numerous accomplishments, including academy elections and breakthrough prizes, but chose to focus on her scientific contributions.

Transition to Dr. Doudna’s Presentation:
Dr. Black transitioned to Dr. Doudna’s presentation, highlighting her work leading up to her current research, which the audience was most familiar with.

Jennifer Doudna’s Background:
Jennifer Doudna is a renowned scientist who has made significant contributions to RNA biology and the discovery of CRISPR-Cas9. She earned her PhD at Harvard Medical School and conducted postdoctoral research at the University of Colorado Boulder. In 1994, she established her own lab at Yale University, where she determined the structure of the P4-P6 domain of group 1 intron. In 2002, she moved to the University of California, Berkeley, where she continued her studies on ribozymes and RNA biology.

Jennifer Doudna’s Research on CRISPR-Cas9:
Jennifer Doudna and her lab initially focused on type 1 CRISPR systems, studying the multi-subunit cascade complex involved in DNA recognition and cleavage. Later, they shifted their attention to type 2 CRISPR systems, where they discovered the Cas9 protein. In collaboration with Martin Yinnick and Emmanuel Charpentier, they developed biochemical assays for Cas9 and demonstrated its ability to cleave DNA with high specificity. In 2012, they published a landmark paper describing the use of Cas9 as a programmable nuclease, which revolutionized molecular biology.

The Impact of CRISPR-Cas9:
The discovery of CRISPR-Cas9 as a genome editing tool had a profound impact on biological research. It allowed scientists to make precise changes to DNA in a wide variety of organisms. In 2013, several labs, including Jennifer Doudna’s, showed that Cas9 could be used to target and cleave specific DNA sequences in mammalian cells. This led to an explosion of research and development in the field of genome editing, with CRISPR-Cas9 becoming a standard tool for genetic engineering.

Jennifer Doudna’s Ongoing Research and Advocacy:
Jennifer Doudna and her lab continue to study the basic mechanisms of CRISPR-Cas9 and other CRISPR systems. They have also been involved in developing new applications for CRISPR-Cas9 technology, including its use in diagnostics and therapeutics. Jennifer Doudna has also been a vocal advocate for responsible and ethical use of genome editing technology. She has called for public discussion and engagement to ensure that CRISPR-Cas9 is used for the benefit of humanity and not for harmful purposes.

Introduction:
Jennifer Doudna highlights the remarkable advancement in gene editing technology, which allows precise changes to DNA sequences in cells. The concept of making targeted changes to DNA, specifically single base pairs, was once considered science fiction.

Inspiration from Bacterial Immune Systems:
Doudna’s research focused on understanding how bacteria defend against viral infections. Curiosity about this biological phenomenon led to the discovery of CRISPR systems.

Presentation Outline:
Doudna’s presentation covers three main aspects: The journey from studying obscure biology to developing a groundbreaking biotechnology. The molecular mechanisms underlying CRISPR technology. Ethical and societal considerations surrounding the use of CRISPR technology in clinical and research settings.

CRISPR Systems and Jillian Banfield’s Contribution:
Doudna’s attention was drawn to CRISPR systems about 10 years ago. Her colleague, Jillian Banfield, a renowned scientist at UC Berkeley, played a significant role in this discovery.

Background and Jill Doudna’s Involvement:
Jill Doudna’s lab was contacted by Jennifer Doudna about unusual sequence signatures found in bacterial genomes, consisting of repetitive DNA sequences and viral DNA fragments. These signatures were associated with CRISPR (clusters of regularly interspaced short palindromic repeats) and Cas genes, suggesting a potential acquired immune system in bacteria.

CRISPR-Cas as an Adaptive Immune System:
The CRISPR-Cas system is a defense mechanism in bacteria against viral infections. It involves the integration of viral DNA fragments into the bacterial genome, creating a genetic vaccination card. When exposed to matching viral DNA, the CRISPR-Cas system uses RNA-guided Cas proteins to target and cut up the viral DNA.

Type 1 CRISPR Systems:
Doudna’s lab initially focused on type 1 CRISPR systems, which involve multiple Cas proteins in the targeting complexes.

Collaboration with Emmanuel Charpentier and Discovery of Cas9:
Doudna met Emmanuel Charpentier, who was studying a different type of CRISPR system in Streptococcus pyogenes. This system had only one Cas protein, called Cas9, which was shown to be important for acquired immunity in this bacterium.

Biochemical Dissection of Cas9:
Martin Jinek and Christoph Chylinski investigated the function of Cas9. They discovered that Cas9 binds to double-stranded DNA, opens it locally, and makes a blunt double-stranded DNA break. This break is determined by a 20-nucleotide sequence in an RNA molecule derived from the CRISPR locus.

Development of the Single Guide RNA System:
Jinek trimmed away extraneous sequences from the two RNA molecules required for Cas9 function, creating a single guide RNA. This simplified system allows for easy manipulation of the DNA cleavage site by changing the RNA sequence in the single-guide construct.

Testing the Single Guide RNA System:
Experiments confirmed that the single-guide RNA system could effectively target and cleave DNA at specific sites. This marked a significant milestone in the development of CRISPR-Cas9 as a versatile gene-editing tool.

Cas9 Protein Structure and Mechanism:
A 3D printed model of the Cas9 protein shows its cleft structure, where the guiding RNA binds. The RNA guides the Cas9 protein to the target DNA site, where it makes a double-stranded DNA break.

How CRISPR-Cas9 Works:
CRISPR-Cas9 is a molecular scalpel that can precisely cut DNA. It consists of a protein complex that forms a helical interaction with one strand of DNA and cuts it. The other strand of DNA is opened up, and two molecular blades cut the DNA.

Genome Engineering with CRISPR-Cas9:
Double-stranded DNA breaks induced by CRISPR-Cas9 can be repaired by two pathways: non-homologous end joining and homologous recombination. By controlling where DNA breaks are induced, precise changes can be made to the DNA of a cell.

Limitations of Protein-Based Genome Editing Technologies:
Protein-based technologies like zinc finger nucleases and homing endonucleases require significant protein engineering. A new protein must be made for each experiment in a cell.

CRISPR-Cas9 Advantage:
CRISPR-Cas9 uses a single protein whose DNA cleaving activity and specificity can be controlled by simply changing a short guide RNA sequence. This allows for easy and rapid changes to the DNA of a cell.

CRISPR-Cas9 in Action:
CRISPR-Cas9 can search through highly packaged DNA to find a single site that matches the sequence of the guide RNA. It unwinds the DNA, forms an RNA-DNA hybrid, and cuts the DNA. Repair enzymes in the cell then repair the cut, potentially integrating new genetic information at the site of the break.

Broad Applications of CRISPR-Cas9:
CRISPR-Cas9 works across different cell types, tissues, and whole organisms. Its ability to precisely change DNA sequence has opened up new avenues for research and therapeutic applications.

Why CRISPR-Cas9 Took Off Quickly:
The power of base pairing allows for RNA-DNA hybridization specificity. It’s a relatively simple way to program a protein for targeting specific genetic sequences.

Applications of CRISPR-Cas9:
Enables various applications, including gene editing, transcriptional control, and multiplex targeting. Can be used to study gene function and develop new therapies for genetic diseases.

Cas9 Modifications:
Easily modified, allowing for different versions with unique properties. dCas9: Deactivated form of Cas9 for transcriptional control without permanent genome changes. Chimeric Cas9: Linked with other enzymatic activities for specific DNA modifications.

Multiplex Targeting:
Naturally multiplex, allowing protection against different viruses in bacteria. Scientists can harness this feature for simultaneous editing at multiple sites in the genome.

DNA Curtains Experiments:
Collaboration with Eric Green’s lab to study Cas9’s search mechanism using DNA curtains. Curtin system visualizes single DNA molecules and protein interactions. Findings revealed: DNA binding begins at PAM motifs, followed by interrogation of adjacent DNA for a match. Binding to PAM triggers a protein structural change and DNA unwinding. Cas9 remains tightly associated with DNA even after cutting.

Cas9 Spends More Time on G-Rich DNA Regions:
Single-molecule experiments suggested that Cas9 spends more time on G-rich DNA regions due to potential PAM motifs and interactions with those sites.

Biochemical Experiment to Test PAM Motif Interactions:
A DNA molecule with a PAM site matching a guide RNA sequence was used in an experiment. The DNA was radiolabeled and had a target site with a PAM. Unlabeled competitor DNAs with varying numbers of PAMs but no complementarity to the target site were added.

Competition Experiment Results:
The cleavage rate decreased as the concentration of competitor DNA with more PAMs increased. The more PAMs in the DNA sequence, the greater the ability of the competitor DNA to prevent binding to the substrate by competition.

Conclusion:
These results indicate that Cas9’s interaction with DNA is primarily driven by PAM motifs rather than the initial recognition of the target site.

Conformational Changes of Cas9:
Cas9 undergoes significant conformational changes upon binding to guide RNA and DNA. A movie created using crystal structures shows the morphing between different structural states of Cas9. The guide RNA binding opens a channel where the RNA-DNA hybrid forms, and a final conformational change allows DNA cleavage. Conformational changes were detected using fluorescence resonance energy transfer between dyes on the protein surface.

Model for Cas9-DNA Interaction:
Cas9-RNA complex has rapid binding kinetics with DNA, slowing down at PAM sequences for interrogation. Only upon sequence complementarity is the interaction stabilized, leading to DNA unwinding, RNA-DNA hybrid formation, and DNA cleavage.

Super Resolution Microscopy of Cas9 in Live Cells:
Spencer Knight used super resolution microscopy to study Cas9 behavior in live cells. Cas9 was labeled with a halo tag and a fluorophore in living cells for visualization.

Cas9 Behavior in Live Cells:
Cas9 moves incredibly fast inside the nucleus, much faster than histone proteins. Both apo form of Cas9 and Cas9 programmed with a nonsense guide have similar rapid diffusion.

Cas9 Particle Dynamics and Target Recognition:
Cas9 particles exhibit different kinetic behaviors based on target recognition in the genome. Cas9 particles show slower movement and longer retention times at target sites with perfect guide RNA matches. The lifetime of Cas9 on a genuine target site is minutes, while it’s less than a second for sites with mismatches.

Cas9 Access to Different Chromatin Regions:
Cas9 particles access both euchromatic and heterochromatic regions in the nucleus. Cas9 occasionally ventures into heterochromatic regions, demonstrating its ability to interact with DNA even in compacted areas.

Differences Between Cas9 Proteins:
Smaller type 2C Cas9 proteins have reduced helicase activity compared to larger type 2a proteins. Type 2C Cas9 proteins are slower at cutting double-stranded DNA due to their weaker unwinding ability. Engineering smaller Cas9 enzymes with improved helicase activity could enhance their genome editing capabilities.

Challenges in Therapeutic Applications of CRISPR-Cas9:
Delivery of CRISPR-Cas9 components into specific cells and tissues remains a challenge. Precise control over DNA repair mechanisms is necessary for accurate and predictable gene editing. Ethical and societal considerations arise when editing germline cells, such as sperm, eggs, or embryos.

Pre-assembled Protein RNA Complexes for CRISPR-Cas9 Delivery:
Pre-assembling Cas9 protein and guide RNA into a complex with chemical ligands enhances targeted delivery. Protein RNA complexes (RNPs) can be efficiently introduced into immune cells via nucleofection. RNPs have a half-life of 24 hours, minimizing off-target effects. Co-delivery of DNA templates for repair enhances the precision and efficiency of gene editing.

Future Directions:
Ongoing research aims to extend these techniques to therapeutic editing and hematopoietic stem cells.

Ethical Concerns Surrounding Human Germline Editing:
Jennifer Doudna realized the potential ethical implications of CRISPR technology, particularly with the ability to edit the germ lines of organisms and transmit genetic modifications to offspring.

Meeting of Scientists to Address Ethical Considerations:
In January 2014, Doudna organized a meeting in Napa Valley with scientists, including David Baltimore and Paul Berg, who had experience in addressing ethical issues related to molecular cloning.

“A Prudent Path Forward”:
The meeting resulted in a publication in Science Magazine proposing a “prudent path forward” for the responsible use of CRISPR technology. They recommended against clinical applications of human germline editing in human embryos, allowing time for broader and global discussions on ethical implications.

National Academies Involvement:
The National Academies of Science in the US, China, and the UK held a meeting to discuss the global use of CRISPR technology. This meeting aimed to promote beneficial applications while respecting different societal norms and ethical values.

Innovative Genomics Initiative:
Doudna is involved in the formation of the Innovative Genomics Initiative, a UC Berkeley-UCSF collaboration focused on using genome editing for various applications, particularly in human health. The initiative seeks funding from philanthropy donors and companies to support research.

Gratitude and Acknowledgements:
Doudna expressed gratitude to her colleagues, collaborators, and funding sources, including the Gates Foundation, Howard Hughes Medical Institute, and the National Science Foundation. She emphasized the importance of basic science funding and pursuing scientific passions without knowing the exact outcomes.