Overview:
The Lloyd Holly Smith Lecture is an annual event where renowned scientists share their experiences and insights to inspire the next generation of scientific leaders. This lecture honors Lloyd Holly Smith, a distinguished figure in American academic medicine who played a pivotal role in elevating UCSF to a leading medical center.

Holly Smith’s Influence:
Holly Smith’s influence extended beyond UCSF, shaping generations of scientific and medical leaders across the country. He served as a personal friend, mentor, and inspiration to many, including the speaker.

Lecture Dedication:
This year’s lecture honors Holly Smith and his wife, Margaret, who were unable to attend due to Holly’s recent knee replacement surgery. Patti Fitzpatrick, who endowed the lectureship, and her father, Jim Weingarten, who was the speaker’s first department chair and later headed the National Institutes of Health, are also acknowledged for their contributions.

Introducing the Guest:
Warner Green is invited to introduce the esteemed guest, Jennifer Doudna, who will deliver the lecture.

Jennifer Doudna’s Background and Accomplishments:
Earned undergraduate degree from Pomona College and PhD from Harvard University. Conducted postdoctoral research at the University of Colorado. Became an assistant professor at Yale University and joined the Howard Hughes Medical Institute. Moved to Berkeley in 2002 and became a professor of molecular biology, cell biology, and chemistry. Received numerous prestigious awards, including the Waterman Award, Lurie Award, Janssen Award, Breakthrough Prize in Life Sciences, Gruber Award, Mastery Prize, and Gairdner Award. Elected to the National Academy of Sciences, National Academy of Medicine, and National Academy of Inventors, as well as a foreign member of the Royal Society. Named one of Time Magazine’s 100 Most Influential People in the World.

The Genesis of Gene Editing Technology:
Scientists have long sought to make precise changes to the DNA code. Recent advances in technology have enabled gene editing, but early methods were complex and limited. CRISPR technology emerged as a simple, fast, and accessible gene editing tool.

CRISPR Technology: A Serendipitous Discovery:
CRISPR technology originated from research on how bacteria fight viral infections using small RNA molecules. Jill Banfield’s computational biology research revealed repetitive DNA sequences in bacterial genomes, suggesting an adaptive immune system. These repetitive sequences, known as CRISPRs, were found to contain DNA derived from viruses that infected the bacteria. CRISPR-associated (Cas) genes were identified near the CRISPR sequences, indicating a potential system for fighting viruses.

Investigating the Role of CRISPR Sequences at the RNA Level:
Jennifer Doudna and Jill Banfield hypothesized that CRISPR sequences might be utilized as RNA molecules. This hypothesis led to further research to understand the function of CRISPR-Cas systems in bacteria.

Background:
Jennifer Doudna discussed the discovery of CRISPR-Cas systems, which are bacterial immune systems used to protect against viruses.

CRISPR Array and RNA Production:
Bacteria with a CRISPR array can defend themselves against viruses by integrating viral DNA into the CRISPR array. These integrated viral DNA segments are transcribed into RNA molecules, forming a long stretch of RNA with multiple virally derived segments.

CRISPR RNA Processing:
A CRISPR system typically has an enzyme that chops the long RNA molecules into functional RNA units. Each functional RNA unit contains one virally derived sequence flanked by identical sequences from the DNA repeats. These identical sequences act as handles for assembling with Cas proteins to form surveillance complexes.

RNA-Guided DNA Cleavage:
The RNA-guided surveillance complexes search for DNA sequences matching the viral sequence in the RNA. Upon finding a match, the complexes latch onto the DNA and cut it up, preventing viral replication.

CRISPR System Diversity:
There are two main classes of CRISPR systems: Class 1: Requires multiple proteins to form targeting complexes with RNA. Class 2: Includes a single protein that acts as an RNA-guided enzyme for DNA targeting.

Collaboration and Research Focus:
Jennifer Doudna initially studied type 1 CRISPR systems and later collaborated with Emmanuel Charpentier’s lab to explore type 2 systems. Their research centered on understanding the function of the Cas9 protein, a key component of type 2 CRISPR systems.

Cas9 as a DNA-Targeting Enzyme:
Cas9 is a unique enzyme that recognizes and binds to specific DNA sequences matching a 20-nucleotide sequence present in a CRISPR RNA. CRISPR RNA provides targeting information, guiding Cas9 to the specific DNA molecule it should latch onto.

DNA Cleavage and Viral Defense:
When Cas9 identifies a matching DNA sequence, it unwinds the DNA and cleaves both strands through its two enzyme active sites. This precise double-stranded break in the DNA leads to the destruction of the DNA molecule, providing an effective defense mechanism against viral infections in bacteria.

Molecular Basis of CRISPR-Cas9 DNA Targeting:
Martin Jinek and Christoph Doudna’s research revealed two fundamental aspects of CRISPR-Cas9 function. A second RNA molecule, known as tracer RNA, is required for producing mature CRISPR RNAs and forms a binding site essential for Cas9 protein binding. DNA targeting requires a match to the 20 nucleotides in the CRISPR RNA and an adjacent PAM sequence (GG dinucleotide in Cas9).

CRISPR-Cas9 as a Programmable DNA-Cutting Tool:
By changing the sequence of a short stretch of RNA in the CRISPR RNA, the Cas9 protein can be programmed to recognize and cut specific DNA sequences. The concept of simplifying the CRISPR-Cas9 system by linking the two RNA molecules into a single guide RNA was proposed. The single guide RNA contains the Cas9 binding structure and the targeting sequence, allowing for easy programming.

Experimental Validation of Single Guide RNA-Directed DNA Cleavage:
Martin Jinek created five different single guide RNAs targeting sequences in a circular plasmid DNA. An agarose gel analysis showed specific DNA fragments released after Cas9 and single guide RNA-directed cleavage. This experiment demonstrated the feasibility of using CRISPR-Cas9 as a programmable DNA-cutting tool.

3D Model of Cas9 Protein and its Mechanism:
A 3D printed model of the Cas9 protein, based on crystallographic data, illustrates its structure and interactions with guide RNA and DNA. During DNA targeting, the Cas9 protein unwinds the DNA strands, allowing for RNA-DNA hybridization.

Discovery of Cas9:
CRISPR-Cas9 is a bacterial defense system that protects against viruses. Cas9 is a protein that cuts DNA at specific locations. Cas9 is guided to the target DNA by a short RNA molecule called a guide RNA.

Applications of CRISPR-Cas9:
CRISPR-Cas9 can be used to make precise changes to DNA in living organisms. This has led to a wide range of applications, including: Gene therapy Crop improvement Basic research

Challenges to Using CRISPR-Cas9:
CRISPR-Cas9 can cause unintended mutations in DNA. Delivery of CRISPR-Cas9 to cells can be difficult. CRISPR-Cas9 can be used for unethical purposes.

Ongoing Research:
Researchers are working to address the challenges of using CRISPR-Cas9. This includes developing new ways to deliver CRISPR-Cas9 to cells and to reduce the risk of unintended mutations. Researchers are also exploring new applications for CRISPR-Cas9, such as using it to treat diseases and improve crop yields.

DNA Target Recognition:
Cas9 binds to a matching sequence in DNA and triggers DNA unwinding. RNA-DNA helix formation activates catalytic activities, leading to DNA cutting. DNA unwinding occurs without external energy source.

DNA Curtains Experiments:
Used to study Cas9 interaction with DNA. Cas9 aligns on DNA strands at programmed positions. DNA binding begins at PAM motifs, triggering DNA unwinding and enzyme activation. Cas9 has high affinity for DNA product, resulting in no substrate turnover.

Structural Insights:
Low-resolution electron microscopy revealed conformational changes upon RNA binding. Crystallographic structures show dramatic rearrangement of RNA and DNA within Cas9. Cas9 assumes active structure only when engaged with guide RNA and matching DNA.

How Cas9 Protein Interacts with DNA:
Cas9 protein swings its active site into position to cut DNA when double-stranded DNA is present. The conformational changes in the protein trigger DNA unwinding and provide energy for melting the DNA duplex.

Cas9 Behavior in Single-Molecule Experiments:
Cas9 binds and releases DNA very quickly and randomly, but slows down at PAM motifs to interrogate adjacent DNA sequences. Base pairing between guide RNA and DNA triggers DNA unwinding and activation of catalytic sites for DNA breaks.

Cas9 Behavior in Live Cells:
Cas9 particles move rapidly around the live cell nucleus, interacting with DNA. Particles show slower kinetics when programmed with a guide RNA that has a perfect match to a sequence in the genome, indicating long-term binding. Cas9 search mechanism involves three-dimensional diffusion rather than processive interaction along DNA length.

Cas9 and Chromatin:
Cas9 can take advantage of transient dynamics of chromatin in animal and plant cells due to its ability to bind and release DNA quickly.

Therapeutic Challenges:
Delivery: How to selectively introduce editing molecules into tissues. DNA Repair Control: How to control the way DNA is repaired after it’s cut. Ethical Considerations: Addressing ethical challenges associated with gene editing, particularly in the human germline.

CRISPR-Cas9 for Huntington’s Disease:
Targeting an expanded short triplet sequence in DNA to correct or mitigate the genetic disease. Collaboration with Roche Biopharma and UCSF neurosurgeons to expand research and address other disease targets in the brain.

Overview:
Jennifer Doudna discusses the work of Brett, her colleague, on using RNPs (RNA protein complexes) for delivering CRISPR-Cas9 components into cells. This method involves introducing preassembled Cas9 protein and guide RNA complexes directly into cells, aiming to minimize off-target effects and immune responses. The strategy allows for cell type-specific delivery and can be tested in animal models.

RNP Delivery Approach:
The strategy involves delivering preassembled RNPs into cells instead of using genetic encoding methods or viral vectors. This approach could potentially reduce off-target effects due to the shorter lifetime of RNPs and eliminate the risk of integrating foreign DNA into the cell’s genome. It also allows for engineering cell type-specific delivery, enabling targeted editing in specific cell populations.

Experiment with Neural Progenitor Cells:
Brett purified Cas9 and formed RNPs with guide RNAs. These RNPs were used to edit neural progenitor cells in the laboratory. A reporter mouse model with a red dye gene integrated into the genome was used to visualize the edited cells. By removing specific DNA sequences using Cas9, Brett was able to activate the red dye gene, indicating successful genome editing.

Challenges in Delivering RNPs to Animal Brains:
The main challenge lies in delivering RNPs into the brain tissue effectively. The goal is to develop methods for targeted delivery to specific brain regions while minimizing damage and immune responses.

Conclusion:
The RNP delivery method for CRISPR-Cas9 genome editing offers advantages in terms of reducing off-target effects and immune responses. It enables cell type-specific delivery and can be tested in animal models. Researchers are working on overcoming the challenges of delivering RNPs to animal brains to enable targeted editing in various brain regions.

In Vitro Testing of Modified Cas9:
Modified Cas9 proteins with cell-penetrating peptides (NLS) were created. Assays confirmed that NLS-modified Cas9 had identical DNA cleavage activity to unmodified Cas9 in vitro.

Direct Delivery of Cas9 into Cells:
Direct delivery of unmodified Cas9 into cells without special treatment resulted in minimal gene editing. Direct delivery of NLS-modified Cas9 resulted in significant gene editing, indicating the effectiveness of cell-penetrating peptides.

In Vivo Gene Editing in Mice:
Injections of NLS-modified Cas9 into mouse brains resulted in gene editing in a concentration-dependent manner. Injection volume remained constant while concentration varied, suggesting specific trafficking of editing molecules.

Cell Type-Specific Gene Editing:
Gene editing was observed in hippocampal and striatal neurons, but not in astrocytes. The reason for this cell type specificity is currently being investigated.

Future Research Directions:
Expanding studies to include mouse models of Huntington’s disease. Exploring other aspects of CRISPR biology, including the diverse flavors of CRISPR pathways and their functions.

Phylogenetic Tree of C2C2 Enzyme:
Researchers analyzed different examples of the C2C2 enzyme and its activities through biochemical experiments.

Pre-CRISPR RNA Cleavage:
C2C2 enzyme binds to RNA molecules produced from the CRISPR array and cleaves them precisely to generate guide molecules for target recognition.

Dual RNA Cutting Activities:
C2C2 enzyme possesses two distinct RNA cutting activities: The first activity enables the enzyme to produce its own CRISPR RNAs. The second activity allows it to target and cleave RNA sequences.

RNA Targeting Behavior:
Unlike Cas9, which precisely cuts RNA sequences at specific locations, C2C2 enzyme exhibits a different behavior. Once it finds a target RNA sequence matching its guide RNA, it initiates a general RNA cleavage activity.

Suicide Protein Hypothesis:
C2C2 enzyme may function as a “suicide protein” upon detecting foreign transcripts in a cell. It triggers a general RNA cleavage response, targeting and destroying nearby RNA molecules.

Experimental Demonstration:
A purified version of C2C2 enzyme was used in an experiment with radiolabeled RNA molecules (Substrate A and B). In the presence of matching CRISPR RNA, Substrate A was cleaved at multiple sites and eventually destroyed. However, when using a non-matching CRISPR RNA, Substrate B remained intact. Adding a small amount of unlabeled RNA A (matching the CRISPR RNA sequence) activated the enzyme, leading to the cleavage of Substrate B despite its lack of matching sequence.

CRISPR-Cas System Applications:
Detecting transcripts in mixtures using a commercially available kit. Detecting ribonuclease using a quenched dye pair in RNA that releases fluorescent signals when cut.

RNA Detection Using CRISPR-Cas:
Mitch O’Connell and Alex Soletsky tested CRISPR-Cas in complex RNA mixtures. Detection was highly specific for the target matching the CRISPR RNA. Detection limit of one picomolar of RNA without PCR amplification.

Diagnostic Applications:
CRISPR-Cas as a simple diagnostic method is being explored. Potential use in marking or visualizing RNA transcripts inside cells.

Mechanism of CRISPR-Cas Enzymes:
CRISPR-Cas enzymes produce their own guide RNAs. A separate RNA activity cuts up transcripts after being activated by detecting a particular RNA sequence.

Future Applications:
CRISPR-Cas technology has opened up new possibilities for genome editing. Ongoing research seeks to address ethical, societal, and environmental implications of CRISPR-Cas applications.

Applications Beyond Medicine:
CRISPR is being utilized in various organisms, from plants and fungi to animals, enabling genetic manipulation and study of previously inaccessible systems. An example is the manipulation of wing patterns in butterflies, providing insights into the genetics behind these patterns.

Mushroom Gene Editing:
U.S. Department of Agriculture does not require GMO labeling for mushrooms genetically modified through CRISPR-based knockout of browning genes. This decision has sparked debate due to varying definitions of GMOs worldwide, leading regulatory agencies to grapple with how to address such agricultural applications.

Germline Editing:
CRISPR-Cas9 technology can be applied to germline cells, potentially altering the genetic makeup of future generations. This raises ethical concerns regarding the use of germline editing for disease mitigation or enhancement purposes.

Collaborative Efforts:
Jennifer Doudna acknowledges the contributions of her colleagues and collaborators in advancing CRISPR research and applications. Funding from organizations like the National Institutes of Health and the National Science Foundation has been crucial in supporting these efforts.

Regulation of C2C2 Protein:
A question is raised regarding the regulation of the C2C2 protein in bacteria, which needs to be specific to its target to prevent self-destruction. Jennifer Doudna responds that this regulation is an active area of research and exploration.

Regulation of CRISPR/Cas9 in Bacteria:
Researchers are investigating the regulation of CRISPR/Cas9 systems in bacteria. Overexpression of these proteins in bacteria leads to sickness or death, suggesting a need for controlled expression. The physiological relevance of CRISPR/Cas9 expression levels and their impact on cells are being studied.

Spacer Acquisition in CRISPR/Cas9 Systems:
The mechanism by which CRISPR/Cas9 systems acquire spacers from RNA viruses or transcripts is not yet fully understood. Researchers are exploring how these systems integrate the sequence used to generate RNA into DNA at the level of DNA.

PAM Sequences and Avoidance of Self-Targeting:
PAM sequences play a crucial role in preventing self-targeting of the CRISPR/Cas9 system in bacteria. In the bacterial chromosome, newly integrated viral sequences are flanked by repeat elements that lack PAMs, making them invisible to the system.

Duration and Dynamics of CRISPR/Cas9 Immunity:
Studies have shown that bacteria actively acquire and lose spacers over time, resulting in dynamic maintenance of CRISPR/Cas9 immunity. The polarity of CRISPR arrays, with new spacers being acquired at one end and lost from the other, suggests a controlled process. The mechanisms underlying the length control and loss of spacers in different bacteria are not yet fully understood.

RNPs for Targeted Gene Editing:
Unlike viral vectors, RNP-based gene editing doesn’t introduce foreign DNA, reducing the risk of integration events and spurious outcomes. RNP particles have a short lifetime, typically around 24 hours, which limits off-target effects and reduces the duration of editing molecule expression. Ongoing research aims to develop RNPs with tissue-selective delivery, allowing for specific targeting of desired cell types.

Ethical Considerations:
Jennifer Doudna’s views on the ethical implications of gene editing have evolved over time. Certain absolute limits exist for gene editing applications, such as germline editing in humans.

Germline Editing: From Skepticism to Practical Considerations:
Jennifer Doudna initially had reservations about germline editing for clinical use due to safety concerns, potential unnecessary applications, and ethical dilemmas.

Israel’s Government-Funded IVF and Pre-Implantation Diagnostic Selection:
In Israel, the government supports couples undergoing IVF and pre-implantation diagnostic selection to screen for genetic defects in embryos. This precedent raises the question of whether germline editing could be permitted to introduce beneficial or healthy changes in embryos.

Potential Cost-Effectiveness and Practicality:
The possibility of germline editing to prevent chronic debilitating diseases could lead to cost savings for healthcare systems. The technology is advancing rapidly, with companies and countries exploring clinical applications.

Four Countries Allow CRISPR-Cas9 Gene Editing in Human Embryos for Research:
The increasing acceptance of CRISPR-Cas9 gene editing in human embryos for research purposes suggests a growing trend towards clinical use.

Post-Translational Modifications of Cas9 Proteins:
There is currently limited knowledge about post-translational modifications of Cas9 proteins.

DNA Template Co-Delivery for Homology-Directed Repair:
Co-delivering templates for repair along with CRISPR-Cas9 can achieve high levels of homology-directed repair in laboratory settings. Nucleofection is one method for co-delivery, but clinical translation requires further research.

Base Pairing Interactions and AAV Vectors for Template Delivery:
Exploring base pairing interactions between template DNA and guide RNA may enable non-covalent association for co-delivery. AAV vectors could also be used for template delivery in clinical settings.

Single-Stranded DNA Oligonucleotides for Homology-Directed Repair:
Research by Geraldine Seydoux at Johns Hopkins shows promising results using short single-stranded DNA oligonucleotides for homology-directed repair.

Overall Perspective:
Jennifer Doudna’s views on germline editing have evolved, recognizing the practical and ethical challenges while acknowledging the potential benefits and inevitability of its clinical applications.