Introduction of the King Lectures:
Clare Hall established the King Lectures in 2016 with a generous donation from Donald West King, a pathologist from Yale who spent time at Clare Hall as a visiting fellow and life member. The lectures have become an important part of the college’s intellectual life, attracting distinguished speakers.

Professor King’s Vision:
Professor King’s vision for the lectures included inviting Professor Doudna to speak, as her name was mentioned in the original concept discussions.

Arranger of this Year’s Lecture:
Dr. Laurie Passmore, a group leader at the Medical Research Council Laboratory of Molecular Biology in Cambridge and a fellow of the Royal Society, arranged this year’s lecture.

Introduction of Professor Doudna:
Dr. Passmore introduced Professor Jennifer Doudna, the Lee Keshing Chancellor’s Chair in Biomedical and Health Sciences and a professor at UC Berkeley.

Professor Doudna’s Research Interests:
Professor Doudna’s research focuses on studying how RNA controls cellular function, particularly how RNAs control genetic information.

Education and Early Career:
Professor Doudna obtained her PhD from Harvard University, did her postdoc at the University of Boulder, Colorado, and started her own lab at Yale University.

Contributions to RNA Research:
During her early career, Professor Doudna made significant contributions to the understanding of catalytic RNAs, including determining structures of RNA and showing how they can catalyze reactions like proteins.

Continued Research at UC Berkeley:
Since moving to UC Berkeley in 2003, Professor Doudna has made essential contributions to understanding RNA and RNA-protein complexes, particularly in regulating gene expression.

Serendipitous Beginnings:
Jennifer Doudna’s journey with CRISPR began in 2007 when she collaborated with Jill Banfield, a scientist studying bacteria through bioinformatics. Banfield discovered curiously repetitive DNA elements in bacterial genomes that appeared to adapt over time. This led to the hypothesis that these CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) might be part of an RNA-driven adaptive immune system in bacteria.

Unlocking the CRISPR Mystery:
Doudna teamed up with Emmanuel Charpentier to investigate the mechanism of CRISPR-Cas9, a part of the bacterial defense system that cuts up foreign DNA. They discovered that CRISPR-Cas9 could be harnessed to precisely edit DNA in any cell, including human cells, opening up vast possibilities for genetic research and therapy.

CRISPR-Cas9 Technology:
The CRISPR-Cas9 system utilizes a guide RNA molecule to direct the Cas9 protein to a specific DNA sequence, where it can precisely cut and modify the DNA. This technology has revolutionized genetic engineering, enabling researchers to make precise changes to DNA with unprecedented ease and efficiency.

Therapeutic Promise:
CRISPR-Cas9 has shown immense potential in the field of human health, offering new avenues for treating genetic diseases and developing innovative therapies. Doudna highlighted the exciting advancements in genome editing for therapeutics, marking a new era of possibilities in treating various diseases.

Beyond Medicine:
CRISPR-Cas9 technology has applications beyond medicine, including in agriculture, biotechnology, and environmental science. Doudna emphasized the diverse opportunities that CRISPR presents, extending its potential to solve some of humanity’s greatest problems.

Collaboration and Curiosity:
Doudna emphasized the importance of curiosity-driven science and collaboration in scientific discoveries. She highlighted the role of serendipity and collaboration in her own journey with CRISPR, which led to the development of a groundbreaking technology.

CRISPR: A Bacterial Immune System:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an adaptive bacterial immune system that protects against viruses (bacteriophage). CRISPR captures segments of foreign DNA and inserts them into the CRISPR locus, creating a record of past infections. RNA guides produced from the CRISPR locus direct CRISPR-associated proteins to identify and cleave matching foreign DNA.

CRISPR-Cas9 for Genome Editing:
CRISPR-Cas9 can be harnessed for genome editing in eukaryotic cells, such as animal, plant, and human cells. The RNA guide directs the CRISPR-Cas9 complex to a specific DNA sequence, where it introduces a double-stranded break. The cell’s DNA repair machinery can then be used to modify the DNA sequence at the break site.

Advantages of CRISPR-Cas9 for Genome Editing:
CRISPR-Cas9 is an RNA-guided system, making it easy to change the target DNA sequence by altering the RNA guide. CRISPR-Cas9 is more efficient and easier to implement compared to previous genome editing technologies.

Historical Context:
The use of double-stranded DNA breakage for genome editing has been studied for some time. CRISPR-Cas9 stands out due to its RNA-guided nature, which simplifies the process of targeting specific DNA sequences.

RNA-Guided CRISPR-Cas9 System:
CRISPR-Cas9 is a natural two RNA-guided system that targets DNA. One RNA, called the tracer, forms a molecular structure that enables interaction with DNA.

Double-Strand Breaks and PAM Sequence:
When the system interacts with DNA, it generates double-strand breaks. The system requires a PAM sequence, a specific motif in the DNA adjacent to the target, for targeting. The PAM sequence allows the immune system to distinguish between foreign and self DNA.

Linking the Two RNAs:
In nature, CRISPR-Cas9 is a two RNA-guided system. Researchers realized that the two RNAs were likely close together and could be linked together. By creating a continuous strand of RNA with the guide sequence on one end and the recognition part for Cas9 on the other, they simplified the system.

Experiments to Test Programmable DNA Cleavage:
Jennifer Doudna and Martin conducted experiments to test the programmability of the Cas9 system in guiding RNA-directed DNA cleavage.

Single vs. Two-Component Guide RNA Systems:
They explored two formats of guide RNAs: one with a separate RNA tracer and another with a single-form guide RNA containing both the targeting sequence and the assembly mechanism.

Visualization of DNA Cleavage:
Using electrophoretic gel systems, they observed DNA cleavage when guide RNAs were added to the reaction, resulting in faster migration of the cleaved DNA fragments.

Programmability of Cas9:
By designing guide RNAs with different targeting sequences, they demonstrated precise cleavage of DNA at specific sequences.

Confirmation of Programmable DNA Cleavage:
In a plasmid DNA experiment, they showed that different guide RNAs led to distinct DNA fragments with varying mobilities on the electrophoretic gel, confirming the programmability of Cas9.

Two-Component System and Reprogramming:
The single-molecule format of the guide RNA allowed for easy reprogramming and redeployment of the Cas9 system to target different DNA sequences.

Significance of the Findings:
These early experiments provided strong evidence for the programmability of Cas9, paving the way for its development as a powerful gene-editing technology.

CRISPR Mechanism for Genome Editing:
CRISPR-Cas9 system uses nuclear localization signals to guide RNA molecules into the nucleus of cells. The RNA guides search for specific DNA sequences that match their own sequence. Upon finding a match, the Cas9 protein melts open the DNA and forms an RNA-DNA helix, triggering DNA cleavage.

Repair of Broken DNA Ends:
Repair enzymes in the cell detect the broken DNA ends and seal them up by making small changes in the DNA sequence. Alternatively, a DNA template can be provided for repair, allowing insertion of new genetic sequences at the break site.

Applications of CRISPR:
Gene disruptions and replacements. Control of gene transcription (output). Detection of RNA or DNA molecules for diagnostic purposes. Imaging of specific sequences within the genome.

Global Impact of CRISPR:
Widely adopted for research, medical, and agricultural applications. Potential for targeted modifications in plants, leading to significant agricultural advancements.

Ethical Considerations:
Early recognition of potential risks associated with CRISPR technology. Concerns raised about the use of CRISPR to modify embryos, particularly in humans.

Initial Discovery of Heritable Genome Editing in Monkeys:
CRISPR-edited monkey embryos were implanted, resulting in offspring with modified DNA transmissible to future generations. The realization of heritable genome editing in humans became a possibility.

Ethical Challenges and the Innovative Genomics Institute:
The potential ethical challenges of heritable genome editing in humans sparked discussions among experts. The Innovative Genomics Institute was established to address these challenges.

International Meetings and Reports:
International meetings involving scientific academies delved into the current state of CRISPR science and its legal, societal, and ethical aspects. These meetings led to reports examining various considerations for using CRISPR in heritable genome editing.

Unethical Use of CRISPR in Human Embryos:
A scientist announced the use of CRISPR in human embryos, resulting in the birth of girls with CRISPR-modified DNA. The international backlash condemned the unethical nature of this work, leading to the scientist’s imprisonment.

Current Focus on Somatic Cell Editing:
The majority of clinical development involving CRISPR focuses on somatic cell editing. Somatic cell editing targets cells in individuals, impacting their health or disease state, but not affecting future generations.

Introduction:
CRISPR technology has the potential to treat genetic diseases like sickle cell disease by making corrective changes to the genome of patients.

Background:
Sickle cell disease is caused by a single base pair change in one gene, leading to a mutated form of beta globin. This mutation results in sickled red blood cells, causing organ failure and pain crises. Traditional treatments for sickle cell disease are palliative, such as blood transfusions, but do not provide a cure.

CRISPR as a Potential Treatment:
CRISPR technology can target the source of the disease by making corrective changes to the genome. By doing this, patients can produce normal red blood cells and potentially be cured of sickle cell disease.

Biology of Hemoglobin Production:
During fetal development, gamma globin is produced as the fetal form of hemoglobin. After birth, gamma globin production decreases, and beta-globin, the adult form of hemoglobin, is turned on.

Sickle Cell Disease Onset:
In patients with two copies of the sickle cell gene, sickle hemoglobin starts to be produced within three months after birth. This leads to the onset of sickle cell disease symptoms.

Research on Hemoglobin Regulation:
Scientists have studied the biology of hemoglobin regulation, including the role of transcription factors like VCL 11 a. This research has provided insights into the mechanisms of hemoglobin production and the potential for targeting these mechanisms for treatment.

Background:
Sickle cell disease is a genetic disorder caused by a mutation in the gene that produces hemoglobin, the protein that carries oxygen in red blood cells. This mutation causes red blood cells to become sickle-shaped, which can block blood flow and cause pain, tissue damage, and organ failure. There is no cure for sickle cell disease, but treatments can help manage the symptoms.

CRISPR-Cas9 Therapy:
CRISPR-Cas9 is a gene-editing technology that can be used to correct the mutation that causes sickle cell disease. CRISPR-Cas9 works by using a guide RNA to target the mutated gene and a Cas9 protein to cut the DNA at that location. Once the DNA is cut, the cell’s natural repair mechanisms can be used to insert a correct copy of the gene into the genome.

Clinical Trials:
Clinical trials of CRISPR-Cas9 therapy for sickle cell disease have shown promising results. In one trial, 15 patients with sickle cell disease were treated with CRISPR-Cas9 therapy. After one year, all 15 patients had normal levels of fetal hemoglobin, which is a type of hemoglobin that is produced during fetal development and is resistant to sickling. The patients also had significant improvements in their symptoms and quality of life.

Future Directions:
Researchers are working to improve CRISPR-Cas9 therapy for sickle cell disease and to make it more widely available. One goal is to develop a way to deliver CRISPR-Cas9 therapy to patients without the need for a bone marrow transplant. Another goal is to reduce the cost of CRISPR-Cas9 therapy so that it is more affordable for patients.

Development of In Vivo Genome Editing:
In vivo genome editing is necessary for treating diseases where cells cannot be easily extracted, edited, and reinserted into the patient. Conditions like cystic fibrosis, diabetes, Huntington’s disease, and muscular dystrophy are examples where in vivo editing would be beneficial.

Current Status of CRISPR-Based Clinical Trials:
Approximately 250 patients have been treated with CRISPR in clinical trials. Information about these trials can be found on clinicaltrials.gov, a US National Institutes of Health website.

Challenges in Expanding CRISPR Therapies:
To reach tens of thousands of patients, researchers need to improve the methods for delivering CRISPR editors in vivo.

Example of In Vivo Genome Editing Trial:
Intelia, a company focused on developing CRISPR-based therapies, announced a trial involving in vivo editing of hepatocytes (liver cells) to treat a rare genetic disorder. This trial demonstrates the potential of in vivo genome editing for treating diseases.

CRISPR-Cas9 for Liver Diseases:
CRISPR-Cas9 was used to edit liver cells in patients with TTR amyloidosis, a rare genetic disorder leading to liver destruction. CRISPR molecules were delivered through a one-time injection using lipid nanoparticles, similar to mRNA COVID-19 vaccines. The same delivery method could be used for different genetic disorders by changing the guide RNA.

CRISPR for Sickle Cell Disease:
Current treatment for sickle cell disease involves bone marrow transplantation, which is lengthy, costly, and requires hospitalization. Scientists are exploring alternative delivery methods, such as lipid nanoparticles or chemically modified Cas9, to directly inject CRISPR molecules into patients, reducing treatment duration and cost.

Viral Vectors for Cell-Specific Targeting:
Research is ongoing to harness the cell-infecting capabilities of viruses for CRISPR delivery. Modified viruses can specifically target desired cell types and deliver CRISPR molecules for genetic editing.

Genome Editing Delivery via Viral Packaging:
Jennifer Doudna and her team explored delivering pre-assembled Cas9 guide RNA complexes using viral packaging. The goal was targeted genome editing in vivo by programming particles to deliver editors to specific cells. They achieved exciting results and are working on the structural biology of these particles.

Addressing Challenges in Genomic Medicine Access:
Melinda Kliegman led a task force to investigate solutions for genomic medicine access. They proposed a dynamic pricing structure linked to the GDP of the country. Innovation in manufacturing, licensing of intellectual property, and creative partnerships were identified as key strategies.

Path to an Affordable Cure for Sickle Cell:
The path from ex vivo therapies to an affordable cure involves collaboration among scientists, regulators, companies, and the community. This includes developing better technology, production methods, and regulatory frameworks.

CRISPR’s Impact on Human and Planetary Health:
CRISPR is being applied to address challenges in human health and the health of the planet. The Innovative Genomics Institute focuses on using CRISPR in microbiomes, communities of microbes in our bodies and the environment.

Editing the Human Gut Microbiome and Cow Rumen Microbiome:
CRISPR is used to make targeted changes in the human gut microbiome, linked to diseases like asthma. In livestock, the cow rumen microbiome is edited to reduce methane production, a potent greenhouse gas.

Non-Tenure Track Positions for Collaborative Research:
The Innovative Genomics Institute hired scientists into non-tenure track positions with principal investigator roles, research groups, and funding. This collaborative lab allows scientists to focus on microbiome editing research.

Jennifer Doudna’s Collaborative Lab:
Doudna established a collaborative lab, rewarding teamwork and collaboration to tackle microbiome editing challenges, aiming for real-world solutions.

CRISPR-Cas Proteins:
RNA-guided CRISPR-Cas proteins are valuable for programmable genome editing and genetic manipulations, allowing precise targeting of specific sequences.

Clinical Trials and Safety:
Clinical trials have demonstrated the safety and effectiveness of CRISPR-based therapies, opening the door for expanded use in treating and curing diseases.

Expanding CRISPR’s Use:
The next decade of CRISPR research will focus on expanding its use and developing new applications, benefiting a wider range of patients.

Germline Editing:
In clinical treatments using CRISPR-Cas, germline editing is not observed, ensuring that modifications do not pass on to future generations.

Cattle Microbiome Editing:
Doudna’s team aims to reduce methane emissions from cattle by editing their rumen microbiome, offering a sustainable and affordable solution.

CRISPR in Human Cells:
It is possible to introduce CRISPR mechanisms into human cells to fight viruses, although challenges exist due to the slow growth rate of human cells.

Off-Target Editing:
Off-target editing is a concern in CRISPR therapy, but it is manageable by limiting the amount of editor introduced and using primary cells.

CRISPR and Aging-Related Diseases:
CRISPR is currently more applicable to monogenic disorders, while addressing aging-related diseases, which involve multiple genes, presents a significant challenge.

Hemoglobin Sickle Cell Experiment:
Early CRISPR capabilities focused on DNA cutting and inducing small changes, which led to the initial approach of targeting fetal hemoglobin in sickle cell disease.

CRISPR Gene Editing Approaches for Sickle Cell Disease:
CRISPR can be used to disrupt the production of a repressive protein, leading to fetal hemoglobin production in sickle cell disease. An alternative approach involves correcting the disease-causing mutation by inserting a corrective sequence into the beta globin gene. Ongoing clinical trials, such as the one led by Jennifer Doudna, utilize CRISPR for gene correction in sickle cell disease.

Co-evolution of CRISPR and Cas Proteins:
CRISPR-Cas systems involve RNA-protein complexes that have evolved to work together. In CRISPR-Cas9, two RNAs are used, with the tracer RNA made in a single copy in the genome. The high levels of tracer RNA allow for efficient base pairing with CRISPR repetitive sequences, enabling cleavage of the CRISPR RNA into functional units. Variations in CRISPR-Cas systems exist, with some using a single guide RNA or flipping the orientation of the RNA.

Bottlenecks and Future Directions:
Challenges in scaling CRISPR technology include cost, delivery methods, and potential off-target effects. If unlimited funding were available, research could focus on addressing these bottlenecks, developing more efficient and targeted delivery systems, and exploring novel applications of CRISPR for health and longevity.

Delivery and Targeted Editing:
Delivery is a major bottleneck for clinical applications of CRISPR-based therapies. Current methods are expensive, invasive, and time-consuming. Targeted delivery to specific cells is essential to avoid editing every cell in the body.

Liver as a Target Organ:
Liver is naturally targeted by many substances, including lipid nanoparticles. This makes it a suitable target for CRISPR-based therapies. Other organs, such as blood and the eye, are also being explored for targeted editing.

Maintaining Modified Microbiome:
Editing the microbiome to improve health raises the question of how to maintain the modified microbiome over time. Nutritional approaches, such as providing specific vitamins, are being investigated to sustain the edited microbiome.

Identifying and Addressing Off-Target Effects:
Off-target effects, where CRISPR edits unintended DNA sequences, are a concern in clinical applications. Thorough testing and regulation are necessary to ensure safety and minimize potential adverse effects. Clinical trials and ongoing research aim to identify and address off-target effects.

Epigenome Editing for Reversible Changes:
Epigenome editing, which involves non-permanent changes to DNA, is being explored as a potential approach. Epigenetic modifications can persist over multiple cell generations but are potentially reversible.

Ethical Considerations for Germline Editing:
Modifying the germline (sperm or eggs) raises significant ethical questions. The potential impact on future generations and the uncertainty of long-term consequences present challenges for the application of CRISPR-based therapies to the germline.

Germline Editing: Inevitable and Sooner Than We Think:
Jennifer Doudna believes that germline editing, the modification of human embryos’ DNA, will inevitably occur sooner than expected. Ongoing research in the UK explores human embryo genome editing with CRISPR to understand DNA repair and safe targeting.

Addressing Genetic Diseases:
Doudna emphasizes the rarity of inheriting two copies of a disease-causing gene, making germline editing unnecessary for most genetic diseases. In vitro fertilization and embryo selection can potentially mitigate genetic disorders without germline editing.

Beyond Disease Mitigation:
Doudna anticipates that once germline editing becomes safe and effective, people may seek to use it to enhance their children’s traits, leading to a broader range of applications beyond disease mitigation.

Potential Applications of CRISPR Technology:
CRISPR technology holds immense potential for genetic engineering, including applications in medicine, agriculture, and industry. In medicine, CRISPR can be used to treat diseases by correcting genetic mutations, such as in the case of sickle cell disease or cystic fibrosis. CRISPR can also be used to enhance human traits, such as increasing muscle mass or intelligence, but such applications raise ethical concerns.

Ethical Concerns Surrounding CRISPR Technology:
The use of CRISPR to enhance human traits could lead to a divide between those who can afford the technology and those who cannot, exacerbating social inequalities. CRISPR technology could also be used to create designer babies, where parents select specific traits for their children, which raises concerns about eugenics and the potential for discrimination. There is a risk that CRISPR technology could be used for malicious purposes, such as creating bioweapons or modifying human genes in ways that could have unintended consequences.

Challenges in Delivering CRISPR Technology to Target Cells:
One challenge in using CRISPR technology is delivering the CRISPR components to the target cells. Researchers are exploring various methods for delivery, such as using viral vectors, nanoparticles, or modified proteins. Each delivery method has its own advantages and disadvantages, and researchers are working to develop more efficient and targeted delivery systems.

Addressing the Precision of CRISPR Editing:
Inducing double-strand breaks during CRISPR editing can occasionally lead to genome chromosome loss and other unintended effects. Researchers are working to mitigate these risks by developing new methods for editing genomes without inducing double-stranded breaks. Base editing and prime editing are two such methods that can make targeted changes in genomes without causing double-stranded breaks, but they still have limitations and require further research.