Lecture Overview:
The BEYOND Annual Lecture is a prestigious event where world-renowned scientists are invited to share their insights and visions for the future in their respective fields.

BEYOND Center:
The BEYOND Center for Fundamental Concepts in Science confronts big questions, such as the emergence of meaning and the origin of DNA’s coded information.

ACE Collaboration:
The BEYOND Annual Lecture is co-sponsored by ACE, the Arizona Cancer Evolution Center, which focuses on understanding tumor cell evolutionary dynamics and cancer’s role in shaping life’s evolution.

Speaker Introduction:
The speaker for the evening is Jennifer Doudna, a distinguished professor of chemistry and biochemistry and molecular biology at the University of California, Berkeley.

Gene Editing Expertise:
Dr. Doudna’s pioneering work on gene editing technology has opened up new possibilities and responsibilities in the field.

Expertise and Qualifications:
Dr. Doudna is highly qualified to discuss the significant issues surrounding gene editing, making her an ideal choice for this lecture.

Curiosity-Driven Experiments:
Scientists were curious about how bacteria fight viral infections. Investigations focused on understanding the mechanisms of bacterial immunity.

Unexpected Discovery:
Experiments led to the discovery of a remarkable technology called genome editing. This technology allows for precise editing of the code of life, DNA.

The Power of Genome Editing:
Genome editing offers the ability to change specific genes or DNA sequences. It has wide-ranging applications in various fields, including medicine, agriculture, and basic research.

Implications for Humanity:
Genome editing raises profound questions about its impact on humans and the natural world. Ethical considerations and responsible use of the technology are crucial.

Future Possibilities:
Genome editing has the potential to revolutionize medicine by curing genetic diseases and improving human health. It can also contribute to sustainable agriculture and address global food challenges.

Collaborative Efforts:
The potential benefits of genome editing require collaboration among scientists, policymakers, and the public. The goal is to harness this technology for the betterment of society.

CRISPR Pattern in Bacterial DNA:
Many bacteria possess a unique pattern of repeated DNA sequences interspersed with short unique sequences, known as CRISPRs. These patterns are observed in the circular chromosome of bacteria and consist of identical sequences of about 30 to 40 base pairs flanked by unique DNA. The CRISPRs co-vary with CRISPR-associated (Cas) genes, suggesting a conserved system in bacteria.

CRISPRs as Bacterial Vaccination Card:
Bioinformatics analysis revealed that the unique DNA sequences within CRISPR elements are derived from viruses that infect bacteria. This observation suggested that CRISPRs serve as a bacterial vaccination card, storing pieces of DNA from invading viruses in their own genome.

Hypothesis of Adaptive Immune System:
Jill Banfield hypothesized that CRISPRs might be part of an adaptive immune system, allowing bacteria to acquire immunity to infecting viruses. Bacteria with CRISPR elements can acquire DNA pieces from viruses and generate RNA copies of these sequences. The RNA molecules are processed and combined with Cas proteins to form RNA-guided complexes.

RNA-Guided Complexes and DNA Cleavage:
The RNA-guided complexes search the cell for matching DNA sequences complementary to the RNA guide. Upon finding a match, the Cas protein is recruited to the DNA and cleaves it, destroying the viral nucleic acid. This mechanism provides bacteria with immunity against invading viruses by recognizing and destroying their DNA.

Research Collaboration:
Jennifer Doudna’s lab collaborated with Jill Banfield’s lab to investigate the CRISPR adaptive immune system in bacteria. Doudna’s lab focused on understanding the molecular mechanisms of CRISPR function, while Banfield’s lab continued researching microbes in the environment.

Environmental Sampling and CRISPR Diversity:
Banfield’s lab collected environmental samples, such as groundwater and soil, to study the diversity of CRISPR adaptive immune systems. By analyzing the DNA sequences of microorganisms in these samples, researchers uncovered a wide range of CRISPR pathways.

CRISPR as a Fascinating Research Area:
The diversity of CRISPR systems provides a wealth of opportunities for researchers to study the molecular mechanisms and ecological implications of these adaptive immune systems. The study of CRISPRs offers insights into the mechanisms of immunity, evolution, and the interactions between microbes and their environment.

Diversity of CRISPR Systems:
CRISPR systems are classified into two classes based on their complexity and genetic organization. Class 1 systems require multiple genes for immune system function, while class 2 systems have a single large gene encoding an essential protein.

Collaboration and a Key Question:
In 2011, Jennifer Doudna and Emmanuel Charpentier, a microbiologist, met at a conference and realized they could collaborate to investigate the function of Cas9, a key protein in CRISPR immunity.

Unique Properties of Cas9:
Cas9 intrigued researchers because it seemed to perform complex functions with just one protein, unlike other CRISPR systems requiring multiple proteins. Doudna’s interest was piqued, considering Cas9 a potential “Swiss army knife” with diverse functionalities.

Cas9’s DNA-Cutting Mechanism:
Cas9 acts as a chemical cleaver, inducing double-stranded breaks in DNA at specific sites matching the sequence in the guide RNA molecule. This DNA break triggers destruction of the DNA, helping bacteria eliminate viral invaders.

Engineering the Single Guide RNA:
Martin Jinek, a postdoc in Doudna’s lab, discovered that Cas9 is guided by two separate types of RNA: CRISPR RNA and tracer RNA. By linking these RNAs into a single guide RNA, researchers could easily change the address label to direct Cas9 to any desired DNA segment.

Realizing the Tool’s Potential:
This engineering step transformed the project from a curiosity-driven investigation to a powerful tool for manipulating the code of life in any cell. By altering the sequence in the RNA molecule, researchers could trigger double-stranded breaks at any desired sequence using molecular biology methods.

Prior Research on DNA Repair Pathways:
Concurrently, extensive research over the previous two decades had revealed DNA repair pathways in human, animal, and plant cells. This knowledge was crucial for understanding how cells would respond to Cas9-induced DNA breaks, paving the way for genome editing applications.

Mechanism of CRISPR-Cas9 in Eukaryotic Cells:
CRISPR-Cas9 introduces double-stranded DNA breaks in eukaryotic cells, which are repaired by cellular mechanisms. DNA repair pathways can introduce small changes or insert new DNA sequences at the break site. Cas9 searches through the genome guided by its RNA molecule to find and cut specific DNA sequences.

Rapid Adoption of CRISPR-Cas9 Technology:
Laboratories worldwide quickly adopted CRISPR-Cas9 for genome editing in various organisms. Simplicity, ease of use, and affordability of the technology contributed to its widespread adoption. CRISPR-Cas9 outpaced earlier genome editing technologies due to its adaptability and accessibility.

Exponential Growth in CRISPR-Cas9 Publications:
Since 2012, there has been an exponential increase in scientific publications utilizing CRISPR-Cas9. As of now, there are nearly 13,000 publications across diverse fields of biology using this technology.

Ongoing Research to Understand CRISPR-Cas9:
Researchers aim to gain a deeper understanding of the molecular mechanism of CRISPR-Cas9. Questions regarding DNA recognition, error rates, and cellular repair mechanisms are being explored. 3D models and structural studies help visualize and analyze the interactions between Cas9, RNA, and DNA.

Investigating DNA Recognition by Cas9:
Scientists are studying how Cas9 accesses and cuts DNA precisely. They are examining how Cas9 distinguishes correct from incorrect target sites and how the cell responds to DNA repair. Ongoing research aims to improve the accuracy and specificity of CRISPR-Cas9 for various applications.

Structure of Cas9:
Cas9 is an RNA-guided protein with a unique structure that resembles a clamshell or a baseball glove. It consists of two chemical cleavers that swing into position to cut the DNA strands.

Recognition Mechanism:
Cas9 uses a guide RNA to identify the target DNA sequence. The guide RNA binds to the target DNA, creating an RNA-DNA hybrid. This interaction triggers a conformational change in Cas9, activating the cleavers.

DNA Binding and Cleavage:
Cas9 binds to the DNA and pries apart the two strands, creating a channel down the center of the protein. The cleavers then swing into position and cut the DNA at the desired location. The accuracy of Cas9 is due to evolutionary selection, as bacteria rely on it to protect against viral infection without damaging their own genome.

Energetics of the Process:
The process of DNA binding and cleavage does not require external energy sources like ATP or GTP hydrolysis. The conformational changes in Cas9 and the interaction with the RNA-DNA hybrid provide the necessary energy to melt apart the DNA strands.

Public Health:
CRISPR gene editing has been used to alter the genomes of pigs to make them potentially better organ donors for humans. Gene editing has also been explored as a way to control mosquito populations and prevent the spread of parasites and diseases. These advancements raise important questions about the safety and ethics of using gene editing technology in animals and organisms that may be released into the environment.

Agriculture:
CRISPR gene editing has been used to alter tomatoes to bear much heavier crops without altering other traits, potentially increasing yields. The same gene that affects yield in tomatoes is found in many other plants, raising the possibility of increasing yields in other crops as well. Gene editing could also be used to alter genes in mushrooms to prevent them from turning brown when cut open, potentially making them more appealing to consumers.

Biomedicine:
CRISPR gene editing has been used to alter the genomes of farm animals and animals that are important biomedically, such as pigs. This technology could potentially be used to make animals better organ donors for humans. Gene editing could also be used to create animal models of human diseases, which could help researchers develop new treatments.

CRISPR-Cas9 Regulation:
In the United States, mushrooms edited with CRISPR-Cas9 without foreign DNA introduction are not considered GMOs. In Europe, agricultural products modified with CRISPR-Cas9 are proposed to be classified as GMOs. Regulatory inconsistencies across countries raise questions about the handling and marketing of CRISPR-Cas9 products.

CRISPR-Cas9 in Biomedical Applications:
Researchers are investigating the use of CRISPR-Cas9 to treat neurodegenerative diseases by editing genes in the brain. Experiments in mouse models show promising results for Huntington’s disease treatment. CRISPR-Cas9 can be used to detect infectious bacteria, viruses, antibiotic-resistant organisms, and genetic predispositions. This technology has potential applications in point-of-care diagnostics in resource-limited settings.

Somatic and Germline Editing:
Somatic cell editing makes genetic changes in non-heritable cells, affecting only the individual. Germline editing creates heritable changes that affect an individual and their offspring. Germline editing was demonstrated early in mice, rats, zebrafish, and other organisms. The possibility of germline editing in humans raises ethical and societal concerns.

International Meeting and National Academies of Science Report:
In 2015, a meeting of scientists discussed the implications of germline editing in humans. In 2017, the National Academies of Science published a report on the responsible use of CRISPR-Cas9 technology.

He Jiankui’s Announcement:
Chinese scientist He Jiankui announced in November 2018 that he had genetically edited two baby girls, known as “Lulu” and “Nana,” using CRISPR-Cas9 technology. His goal was to introduce a change in the CCR5 gene to protect the girls from HIV infection. The announcement sparked a global outcry due to ethical concerns and the potential risks of germline editing.

Ethical and Safety Concerns:
The use of germline editing raises significant ethical questions, as it involves making permanent changes to the human genome that can be passed on to future generations. The technology was still in its early stages, and its long-term effects were not fully understood, posing potential risks to the girls’ health and well-being.

Scientific Community’s Response:
The scientific community widely condemned He Jiankui’s actions, expressing concerns about the lack of proper safety protocols, informed consent, and ethical oversight. Calls were made for a moratorium on clinical applications of germline editing until adequate safety and ethical guidelines could be established.

Impact on the Field of Gene Editing:
He Jiankui’s announcement had a profound impact on the field of gene editing, leading to increased scrutiny and regulation of research and clinical applications. It highlighted the need for international collaboration, transparent reporting, and responsible use of gene editing technologies.

Ongoing Discussions and Regulations:
The controversy surrounding He Jiankui’s experiment prompted ongoing discussions and efforts to develop international guidelines and regulations for the ethical and responsible use of gene editing. The incident served as a reminder of the importance of balancing scientific progress with careful consideration of ethical, societal, and safety implications.

Gene Editing in Embryos:
Gene editing in embryos by He Jiankui resulted in genetic changes not previously observed in human populations or tested in animals. This constitutes experimentation on people, raising ethical concerns about the potential consequences of these genetic changes on the health of the baby girls involved.

Hype Surrounding CRISPR Babies:
Media speculation about the possibilities of gene editing to enhance human traits, such as perfect vision, high IQ, and curing baldness, is unfounded. Most of these traits are influenced by complex interactions between numerous genes, making them difficult to alter with gene editing tools.

Future Considerations:
The possibility of germline editing in embryos to make beneficial genetic changes for future generations is on the horizon. Ethical discussions and regulations are urgently needed to address the potential misuse of gene editing technology.

International Forum on Human Gene Editing:
The World Health Organization has convened an international forum to address the ethical and regulatory aspects of human gene editing. Recommendations include establishing a registry for individuals and organizations using germline editing clinically, ensuring transparency and public awareness.

National Academies of Science Initiative:
The National Academies of Science is working on a parallel international forum in collaboration with the WHO group to address gene editing concerns.

Societal and Regulatory Challenges:
Jennifer Doudna highlights the need for clear criteria and requirements for any future clinical use of germline editing in humans. The establishment of such guidelines could provide a framework for regulation and ensure responsible use of the technology.

Applications of CRISPR Beyond Gene Editing:
Doudna emphasizes the diverse applications of CRISPR technology, including gene editing, modulation of protein levels, imaging the genome, and visualizing gene interactions. The versatility of CRISPR has driven scientific advancements and inspired creative approaches in various fields.

Challenges in Delivery and Control:
Doudna acknowledges the importance of efficient delivery methods for gene editing molecules into organisms and tissues. She also highlights the need to address the chemical and societal controls for gene editing, ensuring responsible use and preventing potential misuse.

Ongoing Research and Future Directions:
Doudna recognizes the ongoing fundamental research in CRISPR technology and anticipates future developments beyond CRISPR. She suggests that inspiration for future innovations may come from investigations into the biological pathways of microorganisms by researchers like Jill Banfield.

Collaboration and Funding Appreciation:
Doudna acknowledges the contributions of her team, collaborators, and funding organizations, particularly the Howard Hughes Medical Institute and the National Science Foundation. She expresses gratitude for their support in the early stages of CRISPR research when the potential of the technology was uncertain.

Ethical Considerations in Stem Cell Research:
Doudna comments on the rapidly advancing field of stem cell biology and the potential for organ regeneration using stem cells. She expresses a cautious stance, suggesting that practical applications in this area may still be decades away.

Criticism of Gene Editing in Chinese Twins:
Doudna criticizes the actions of Dr. He, who claimed to be protecting Chinese twins from HIV infection through gene editing, despite established procedures to prevent HIV transmission. She emphasizes the potential downstream consequences and ethical concerns associated with germline modifications.

Worst-Case Scenarios and Responsible Use:
Doudna cautions against the potential negative impacts of gene editing technology, citing examples like gene drives and the CRISPR babies case. She expresses concern that irresponsible use could lead to a backlash against the technology, preventing its beneficial applications.

Conclusion:
Doudna concludes her presentation by acknowledging the profound impact of CRISPR technology and emphasizing the need for responsible use, societal discussions, and ongoing research to ensure the technology’s potential benefits are realized while mitigating potential risks.

Gene Drive Mechanism:
Jennifer Doudna explains the mechanism behind a gene drive, specifically in the context of CRISPR-Cas9. CRISPR-Cas9 is designed to bring along its own guide to recognize the genome and make a cut, enabling efficient editing of any genome it lands in. This allows the trait associated with the gene drive to be inherited and spread rapidly horizontally among organisms.

CRISPR-Cas9 as an Alternative to Antibiotics:
Doudna highlights the growing interest in using CRISPR-Cas9 to replace antibiotics in fighting harmful bacteria. CRISPR-Cas9 can be programmed to target specific bacteria that cause harm to humans. Delivery methods, such as phage or other mobile DNA, can be used to deliver the CRISPR-Cas9 editors to the target bacteria without affecting humans.

Current Research and Future Prospects:
Research in this field is still in its early stages, and it will take some time before CRISPR-Cas9-based treatments can be widely used. At the University of California, Berkeley, a team of microbiologists is actively working on this area of research. CRISPR-Cas9 has the potential to address the growing challenge of antibiotic resistance and provide new strategies for combating harmful bacteria.

CRISPR-Cas9 Effectiveness in Heterochromatin:
CRISPR-Cas9 can edit genes in highly compacted genome regions, such as heterochromatin. The mechanism for this is unknown, but experiments show successful gene editing in heterochromatic regions. CRISPR-Cas9 takes longer to access compacted regions compared to open regions of the genome.

Molecular Movement and Charge Properties:
Protein movement within DNA and the cell nucleus is influenced by chemistry. The charge properties of a protein affect its kinetics and ability to interact in the nucleus. The nucleus is a viscous medium that affects protein movement.

Cushu’s Research on Molecular Movement:
Cushu’s research focuses on how molecules move around DNA and the cell nucleus. He proposes that the charge properties of proteins influence their movement and interactions in the viscous nuclear environment. CRISPR-Cas9 researchers are exploring these ideas to gain a better understanding of molecular movement within the cell.