Background:
Michael Malem, from the School of Immunology and Microbial Sciences, welcomed everyone to the Rosalind Franklin Lecture, a prominent event in the King’s International Lecture Series. King’s Health Partners is a partnership between King’s College London and three South London NHS Foundation Trusts, aiming to integrate excellence and innovation in research, education, and patient care. The Rosalind Franklin Lectures celebrate the importance of fundamental research in understanding and improving human health.

Rosalind Franklin’s Contributions:
Dr. Rosalind Franklin, a biophysicist and crystallographer, is known for her groundbreaking structural studies on DNA, leading to the elucidation of the DNA double helix. Her work has influenced the growth of genomic medicine and other research areas.

Previous Speakers in the Lecture Series:
Randy Sheckman discussed protein secretion autophagy. Stephen Hale described super-resolution fluorescence microscopy and its applications. Roger Chen presented his work on the exploitation of fluorescent proteins to guide surgery.

Introduction of the Guest Speaker:
The speaker for the evening is Dr. Jennifer Doudna from the University of California, Berkeley.

Jennifer Doudna’s Background and Research Interests:
Jennifer Doudna, a chemist and structural biologist, initially focused on topics like catalytic RNAs and RNA silencing. Around 10 years ago, she became interested in CRISPR, a mysterious pathway of bacterial resistance to phage infection.

Collaboration with Emmanuelle Charpentier and Landmark Publication:
Doudna, along with Emmanuelle Charpentier, engineered CRISPR machinery to repurpose it as a tool for targeted alteration of genomic DNA. Their remarkable success was reported in a landmark publication in June 2012, opening the door to numerous applications.

Doudna’s Career Path and Accomplishments:
Doudna is a professor at the University of California, Berkeley, and an investigator with the Howard Hughes Medical Institute. She has received numerous honors and prizes, including the Breakthrough Prize in Life Science and the Heineken Prize for Biochemistry and Biophysics. She is a member of prestigious scientific academies and has been recognized as one of the world’s most influential people and leading global thinkers.

Doudna’s Inspiration from Rosalind Franklin:
Doudna draws inspiration from Rosalind Franklin, a pioneer in DNA research, for her passion, determination, and focus on molecules and structural biology. Franklin’s work laid the foundation for understanding DNA code and replication, while Doudna’s research has led to breakthroughs in DNA editing and manipulation.

Doudna’s Curiosity-Driven Research:
Doudna emphasizes the role of curiosity-driven research and her passion for science since childhood. Her upbringing in a small town in Hawaii without a scientific background highlights the importance of pursuing one’s interests despite challenges. Doudna’s work on CRISPR exemplifies how unexpected directions can lead to groundbreaking discoveries.

DNA as the Information Source for Cells:
DNA contains the chemical information that guides cells in growth, division, and tissue or organism formation. Understanding DNA’s information and manipulating it have been long-standing goals in biology. Scientists have explored methods to copy, sequence, cut, and alter DNA in cells precisely.

Jennifer Doudna’s Initial Perception of RNA:
Initially, Jennifer Doudna viewed RNA as a transient copy of genetic information, primarily serving as a molecule that encodes proteins.

Discovery of Interesting RNA Molecules:
Doudna’s excitement grew when she learned about RNA molecules that performed intriguing functions independently, without encoding proteins. These functional RNA molecules, such as ribozymes, catalyzed chemical reactions at the RNA level.

Structural Studies of RNA:
Doudna’s interest shifted to understanding the structures of functional RNA molecules. She began using X-ray crystallography to solve the structures of RNA molecules.

Move to Berkeley and Focus on Cellular Systems:
Doudna moved to the University of California, Berkeley, seeking opportunities to apply her knowledge of RNA structures to cellular systems. The focus shifted from large RNA molecules to small RNA molecules, particularly microRNAs, which play a role in controlling gene expression.

Collaboration with Jill Banfield:
Jill Banfield, a colleague specializing in metagenomic sequencing, contacted Doudna. Banfield had observed a distinctive pattern of DNA sequences in many bacteria, characterized by repeated elements with unique spacer sequences in between. These sequences, later known as CRISPRs, had a potential connection to RNA, prompting Doudna’s involvement.

CRISPRs as an Adaptive Immune System:
Subsequent bioinformatics research revealed that the spacer sequences in CRISPR arrays often matched sequences found in viruses that infect bacteria. This hint suggested that CRISPRs might represent an adaptive immune system in bacteria.

How CRISPR Systems Work:
CRISPR systems allow cells to detect foreign DNA, steal a piece of it, and integrate it into the CRISPR array. The CRISPR array is transcribed into a precursor CRISPR RNA, which folds into hairpin structures. CRISPR RNAs assemble with Cas proteins to form complexes that search for sequences matching the CRISPR RNA. When a match is found, the Cas enzymes cut the foreign DNA.

Unveiling the Two Classes of CRISPR Systems:
CRISPR systems are broadly classified into two classes: class 1 and class 2. Class 1 pathways involve multiple enzymes assembling into effector complexes with the CRISPR RNA. Class 2 pathways feature a single Cas protein that forms a complex with the CRISPR RNA, simplifying the effector machinery.

Exploring the Diversity of CRISPR Systems:
CRISPR systems exhibit a remarkable diversity in biology, with numerous types and variations. Ongoing research delves into the intricacies of these systems, seeking to uncover fundamental principles with potential technological applications.

Different Types of CRISPR Systems:
Class two systems have a single gene encoding a large protein for protection. These proteins are different from each other and distinct from anything encoded by the genes in class one systems. The evolutionary path of these systems is unclear, but they are diverging quickly in response to phage defense mechanisms.

Jennifer Doudna’s Research on CRISPR Systems:
Doudna’s lab initially focused on class one systems with multiple proteins, allowing for structural studies and cell-based assays. She met Emmanuelle Charpentier, who was studying a class two system in Streptococcus pyogenes, a bacterium causing flesh-eating disease.

Collaboration with Emmanuelle Charpentier:
Doudna and Charpentier’s labs collaborated to investigate the function of the Cas9 protein in the CRISPR system of Streptococcus pyogenes. The collaboration was facilitated by Skype communication between students in the two labs. A serendipitous cultural connection between the Czech and Polish students in the labs sparked the chemistry of the collaboration.

Discovery of Cas9’s Function:
The research team discovered that Cas9 is a remarkable protein capable of cutting double-stranded DNA in an RNA-targeted fashion. A cartoon depicts a double-stranded DNA molecule binding to the Cas9 protein, represented as a blue blob.

Mechanism of Cas9-Mediated DNA Cleavage:
Cas9 forms a complex with two RNA molecules: CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The crRNA guides the Cas9 protein to the target DNA sequence, while the tracrRNA helps in assembling the complex. The Cas9 protein recognizes a specific motif, known as the PAM sequence, adjacent to the target DNA site. Once bound to the target DNA, Cas9 unwinds the double-stranded DNA and forms an RNA-DNA hybrid. The formation of the RNA-DNA hybrid triggers the cleavage of both strands of the DNA by two catalytic domains of Cas9.

Generation of Single Guide RNAs (sgRNAs):
Scientists developed single guide RNAs (sgRNAs) by combining the crRNA and tracrRNA into a single molecule. sgRNAs simplify the programming of Cas9 by allowing researchers to target different DNA sequences simply by changing the 20-nucleotide sequence at the end of the sgRNA.

Energy Source for DNA Unwinding:
Cas9 does not require an external energy source, such as ATP or GTP, to unwind DNA. The mechanism by which Cas9 unwinds DNA without external energy is not yet fully understood. Scientists are studying the structural changes that occur in Cas9 upon binding to nucleic acids to gain insights into the DNA unwinding mechanism.

Structural Changes in Cas9:
Cas9 undergoes dramatic structural rearrangements upon binding to guide RNA and DNA. The binding of guide RNA induces a conformational change that opens up a channel in the protein to accommodate the RNA strand. DNA binding triggers further structural changes, including a rotation of the HNH domain, which is responsible for cleaving the target DNA strand.

Observing Enzyme Activation Conformational Changes:
Previous crystallographic structures of Cas9 revealed an inactive state due to an incorrectly positioned HNH domain. A former graduate student, Sam Sternberg, set up experiments to monitor conformational changes upon RNA and DNA binding using FRET.

Single-Molecule FRET Experiments:
Bulk FRET studies examined the behavior of a mixture of molecules, but single-molecule FRET experiments were desired for more precise information. Collaborations with Ahmet Yildiz and Keith Jung enabled biophysical studies on Cas9 conformational states.

Monitoring Conformational Changes with FRET:
FRET signals were used to monitor conformational changes associated with Cas9 activation. Dye placement on Cas9 allowed for monitoring of different motions.

Binding to DNA Substrates with Different Degrees of Complementarity:
Tethering Cas9-guide RNA complexes to a slide enabled the flow of DNA substrates with varying complementarity. Single-molecule FRET signals revealed conformational changes upon binding to DNA substrates with different mismatches.

Intermediate Conformational State:
Binding to DNA substrates with mismatches resulted in an intermediate FRET state between active and inactive states. This intermediate state suggests a defined mechanism for Cas9 conformational changes.

Improving Cas9 Accuracy:
Collaboration with Keith Jung’s lab led to the design of improved Cas9 enzymes with enhanced accuracy. Mutations based on understanding the conformational changes allowed for increased sensitivity to mismatches.

Future Directions:
Ongoing research aims to further understand the mechanism of Cas9 and utilize this knowledge to improve the technology’s applications.

Background:
Scientists have been studying how cells repair broken DNA to develop methods for precise genome editing. Cells have two primary repair pathways: non-homologous end joining and recombination.

Zinc Finger Nucleases and Talon Proteins:
Zinc finger nucleases and talon proteins were engineered to recognize specific DNA sequences and make precise cuts. These technologies were powerful but required significant protein engineering for each experiment.

CRISPR-Cas9:
Cas9 is a bacterial enzyme that can be programmed to cut DNA at specific sequences by altering the guide RNA that it binds to. This allows for precise genome editing without the need for extensive protein engineering.

Cas9 Mechanism:
In eukaryotic cells, Cas9 searches through DNA in the nucleus to find a match to the guide RNA sequence. When a match occurs, Cas9 opens up the DNA, forms an RNA-DNA hybrid, and makes a double-stranded break. Repair enzymes in the cell then modify or insert sequences at the break site.

Applications of CRISPR-Cas9:
CRISPR-Cas9 was quickly adopted as a genome editing tool in various cell types and organisms. Keith Jung’s lab demonstrated the first experiments in zebrafish, showing precise modifications using this approach.

Gene Editing Applications and Challenges:
Gene editing has potential applications in agriculture and the clinic. Challenges in clinical applications include delivery, controlling DNA repair, ethical issues, and cost.

Delivery of Gene Editing Molecules:
Delivery of gene editing molecules into cells and tissues is a major hurdle. Current approaches are limited and require further development.

Collaboration with Brett Stahl:
Jennifer Doudna collaborated with Brett Stahl to work on Huntington’s disease. Huntington’s disease is caused by a gene with a triplet repeat expansion.

Research Goals:
Doudna and Stahl aimed to use gene editing to correct the triplet repeat expansion. They sought to develop a delivery method for gene editing molecules.

Challenges in Huntington’s Disease Treatment:
Huntington’s disease affects the brain and nervous system. Delivery of gene editing molecules to the brain is particularly challenging.

Introduction of CRISPR-Cas9 into the Lab:
Jennifer Doudna’s lab welcomed a new researcher interested in using RNAi. Doudna suggested an alternative approach that might be more promising. The researcher joined Doudna’s lab and became a driving force behind the development of CRISPR-Cas9 technology.

Challenges of Delivery:
The team faced the challenge of delivering CRISPR-Cas9 into cells, particularly in the brain.

Various Delivery Methods:
Plasmids, viral vectors, and messenger RNA are commonly used for delivery. A novel approach involving direct uptake of assembled Cas9-guide RNA complexes was explored.

Reporter Cell Line for Screening:
A reporter cell line with a gene encoding a red protein (TV tomato) was used for screening. Targeting stop codons with CRISPR-Cas9 could activate TV tomato expression, resulting in red cells.

In Vitro Validation of Assembled Protein-RNA Complex:
Experiments showed that increasing the amount of assembled protein-RNA complex (RNP) introduced into cells led to increased editing. Cell sorting confirmed the correlation between RNP concentration and editing efficiency.

Cell-Penetrating Peptides for Cellular Uptake:
To facilitate cellular uptake without nucleofection, cell-penetrating peptides were appended to Cas9. Experiments demonstrated successful editing in cells with modified Cas9 proteins containing cell-penetrating peptides.

In Vivo Editing in Mouse Brain:
Stereotactic injections of Cas9-guide RNA complexes into the brains of reporter mice were performed. Analysis revealed editing in various types of neurons but not in astrocytes. The reason for this selective uptake is still being investigated, offering potential for tissue-specific targeting.

Ongoing Collaboration and Future Directions:
A collaboration with neurosurgeons at UCSF has been established to further develop this technology. Animal models of Huntington’s disease are being used for further research. The ultimate goal is to conduct clinical trials and utilize this approach for therapeutic applications.

Ethics and Germline Editing:
Jennifer Doudna emphasizes the ethical implications of germline editing, which involves making changes to the entire genome and passing them on to future generations. She highlights the need for responsible use and encourages scientists to engage in discussions about the ethical concerns surrounding germline editing.

Clinical Trials and Applications:
Clinical trials using CRISPR in the immune system are underway in China, combining gene editing with cancer immunotherapy. Doudna mentions the possibility of using CRISPR to correct mutations in developing embryos, raising ethical questions due to its heritable nature.

Antiviral Potential:
Doudna suggests the use of CRISPR systems as antivirals, potentially allowing the excision of integrated viral sequences from genomes. This approach could be valuable as a research tool to study viral manipulation of cells.

Challenges and Research Directions:
Doudna addresses the concern of off-target effects in CRISPR editing and ongoing efforts to minimize them. She highlights the importance of understanding the seed sequence and mismatch effects in reducing off-target activity. Doudna emphasizes the need for more research to identify potential inhibitors of Cas9 and other CRISPR components.

Funding and Support:
Doudna acknowledges the National Science Foundation’s support for her early CRISPR research, emphasizing the significance of small grants in scientific discoveries.

Questions and Answers:
Questions from the audience are addressed, including inquiries about the survival of bacteria during phage infection, resistance to Cas9 in eukaryotic cells, and the relationship between seed sequence and off-target efficiency.

Collaborative Atmosphere at Berkeley:
Doudna highlights the collaborative environment at Berkeley as a key factor in her research success. She emphasizes the importance of collaboration and expertise in cutting-edge biological research.

RNA Targeting CRISPR Systems:
Doudna expresses skepticism about recent reports suggesting that RNA targeting CRISPR systems can work like Cas9 to cut a single transcript. She explains that these enzymes, based on her research, behave more like general ribonucleases, cutting every RNA transcript they encounter.

Translational Aspects of Gene Editing:
Regarding safety and efficacy, Doudna acknowledges the need to balance these factors in both in vivo and ex vivo gene editing methods. She recognizes the potential for personalized medicine, but acknowledges the high cost associated with such treatments, such as a hypothetical $500,000 injection. Doudna emphasizes the need to address accessibility and affordability in order to make these treatments widely available.

Non-Genetic Cas9 Delivery:
Jennifer Doudna highlights the advantages of non-genetic Cas9 delivery methods in enhancing safety by limiting the duration of Cas9 presence in cells, reducing the risk of off-target effects. Pre-assembled Cas9 complexes promote rapid activity while ensuring degradation within 24 hours through natural turnover.

Cost Considerations and Potential Solutions:
Doudna mentions ongoing discussions with pharmaceutical companies regarding the pricing and accessibility of gene editing treatments. Strategies to design sets of guide RNAs that can treat a large number of patients with similar genetic disorders are being explored. The aim is to develop treatments that are effective, affordable, and widely accessible to patients.

Addressing High Costs of Current Treatments:
Doudna expresses concern about the high costs of many current clinical treatments, limiting access for many individuals. She emphasizes the need to work towards making gene editing treatments affordable and available to a broad population.

Striving for Wide Accessibility and Affordability:
Doudna stresses the importance of developing gene editing treatments that benefit a large number of people rather than just a select few. She aims to work towards making these treatments widely accessible and affordable, ensuring equitable access to life-changing therapies.