Jennifer Doudna (UC Berkeley Professor) – CRISPR Biology, A New Era in Genome Engineering (Feb 2016)
Chapters
00:00:06 Precision Medicine: Challenges and Opportunities in Healthcare
Defining Precision Medicine: Precision medicine represents a shift from traditional human judgment (art of medicine) to evidence-based medicine (algorithms designed to improve average outcomes) and now seeks algorithms better than human judgment per individual patient. The question arises of how to measure and ensure confidence in better outcomes for each patient.
Challenges in Precision Medicine: Mathematical models pose a challenge as they are often either too good to be true or too true to be good. Achieving precision medicine requires a delicate balance between accuracy and practicality.
Speaker Introduction: Jennifer Doudna is the invited speaker for the Precision Medicine Data and Discovery for Improved Health and Therapy session and the regulatory RNA session. Questions for Jennifer Doudna will be held during the discussion session at 2.30 p.m. in the same room.
00:03:02 Pioneering Work in RNA Regulation and CRISPR Discovery
Early Life and Education: Jennifer Doudna grew up in Hilo, Hawaii, before pursuing higher education. She completed her doctoral work with Jack Stosek at Harvard and a postdoc with Tom Cech at the University of Colorado Boulder.
Pioneering Work in Ribozymes: Jennifer’s groundbreaking research in engineering and characterizing ribozymes had a significant impact in the field. The findings were taught in undergraduate organic chemistry classes, highlighting their importance.
Academic Career and Research Focus: Jennifer held faculty positions at Yale and Berkeley, where she conducted rigorous studies on the mechanistic basis of RNA regulation. Her work on irises and their role in regulating translation, as well as RNA interference pathways, has garnered recognition and numerous awards.
Collaboration with Jill Banfield and CRISPR Discovery: Jennifer collaborated with Jill Banfield to study a unique microbial community at Iron Mountain with an extremely acidic drainage. They identified curious repeats in microbial genomes, suggesting a record of viral exposures and part of a microbial immune system. Jennifer played a pivotal role in elucidating the CRISPR system and its mechanisms. The discovery of CRISPR led to the development of a groundbreaking tool for manipulating biology.
00:05:02 The CRISPR Revolution: From Discovery to Precision Medicine and Ethical Implications
Background: Jennifer Doudna, a Hilo girl, grew up in Hawaii, where she developed her curiosity about the natural world and her passion for science. The field of precision medicine seeks to connect genotype to phenotype, ultimately aiming to understand the function of specific genetic information in relation to health and disease. In the late 1980s, scientists recognized the potential of making precise changes in DNA at the level of a specific gene or base pair to study gene function and develop new therapies.
CRISPR Discovery and Harnessing: Jennifer Doudna’s lab became involved in precision genome editing technology through curiosity-driven research on the CRISPR bacterial immune system. The CRISPR pathway, initially discovered in bacteria, provides a natural defense mechanism against invading viruses. Understanding the CRISPR mechanism led to its harnessing as a genome editing technology, allowing scientists to make precise changes to DNA at specific locations.
Mechanism and Applications: CRISPR-based genome editing involves a protein called Cas9, guided by a short RNA sequence, which recognizes and cuts specific DNA sequences. By designing the RNA sequence, researchers can target specific genes and introduce precise changes, such as corrections to genetic defects or insertions of new genetic information. This technology has broad applications in basic research, biotechnology, and medicine, including the development of new therapies and the study of gene function.
Ethical and Societal Considerations: The development of powerful genome editing technologies raises ethical and societal questions, including concerns about potential misuse and unintended consequences. Jennifer Doudna emphasizes the importance of responsible and transparent approaches to the development and use of these technologies. Ongoing discussions and collaborations among scientists, ethicists, policymakers, and the public are crucial for addressing these complex issues.
00:10:02 CRISPR-Cas9: A Programmable Genome Editing Technology
Introduction: Jennifer Doudna, a prominent scholar, presents a detailed overview of the CRISPR-Cas9 technology, its discovery, mechanism, and potential applications in genome editing.
CRISPR-Cas9 System: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences are repetitive DNA segments found in bacteria and archaea. CRISPR-associated (Cas) genes are often located near CRISPR sequences and encode proteins involved in the CRISPR-Cas system.
Function of CRISPR-Cas System: The CRISPR-Cas system acts as an adaptive immune defense mechanism in bacteria and archaea. It protects against foreign DNA, such as viral or plasmid DNA, by integrating foreign DNA sequences into the CRISPR locus. These integrated sequences are transcribed into RNA molecules and processed into smaller RNAs. These RNAs form complexes with Cas proteins to target and cleave matching DNA sequences, providing immunity against foreign DNA.
Cas9 Protein: Cas9 is a key protein involved in the CRISPR-Cas system. It interacts with CRISPR RNA (crRNA) and tracer RNA (tracrRNA) to form a complex that binds to and cleaves DNA. The crRNA sequence determines the DNA sequence that will be targeted for cleavage.
Single Guide RNA (sgRNA): Martin Jinek simplified the CRISPR-Cas9 system by linking crRNA and tracrRNA into a single guide RNA (sgRNA) molecule. This sgRNA contains both the targeting information and the Cas9 binding information. The sgRNA can be easily altered to target different DNA sequences.
Genome Editing Using CRISPR-Cas9: The CRISPR-Cas9 system can be harnessed for genome editing by introducing a double-stranded break (DSB) at a specific DNA location. Eukaryotic cells have natural DNA repair mechanisms, such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ can repair the DSB, but it often results in small insertions or deletions at the repair site. HDR can repair the DSB using a donor DNA template, allowing for precise modifications or insertions of genetic information.
Applications of CRISPR-Cas9: CRISPR-Cas9 has revolutionized genome editing and has broad applications in various fields, including: Basic research: studying gene function and regulation. Crop improvement: engineering crops with desirable traits. Medicine: developing gene therapies for genetic diseases. Biotechnology: creating modified organisms for industrial applications.
Conclusion: CRISPR-Cas9 technology offers unprecedented precision and flexibility in genome editing, opening up new avenues for scientific research, medical advancements, and agricultural innovations.
00:21:36 Bacterial CRISPR-Cas9 System: A Versatile Tool for Genome Manipulation
System Advantages and Challenges: Cas9 is a single protein that can be reprogrammed with a guide RNA to target specific DNA sequences, making it adaptable and versatile for various experiments. Protein engineering is a significant challenge for widespread adoption of genome editing technologies, but Cas9 alleviates this issue by enabling easy reprogramming.
Eukaryotic Cell Function: Cas9 searches the entire genome to find a single site matching the guide RNA sequence and forms a complex with the DNA. The complex creates a double-stranded break in the DNA, which is then repaired by the cell’s machinery. This process is remarkable as it works in virtually every cell type tested, including plants, fungi, bacteria, and mammalian systems.
Research and Application: Understanding the basic mechanism of Cas9 is crucial for its application in human cells. Cas9 can be easily modified into a deactivated form (dCas9) that retains DNA binding but lacks cutting activity, enabling applications such as transcriptional control and live cell imaging. The system’s multiplexing capability allows programming Cas9 with multiple guide RNAs to make simultaneous changes to a genome.
Target Recognition Mechanism: Research is ongoing to understand how Cas9 recognizes and targets DNA sequences. CRISPR-Cas systems are naturally multiplexed in bacteria, enabling simultaneous targeting of multiple viruses. This property can be harnessed for experimental purposes to make multiple genomic changes in a single cell.
Conclusion: Cas9 is a powerful and versatile tool for genome editing due to its programmability, ease of modification, and multiplexing capabilities. Its applications range from basic research to potential clinical applications in the near future.
00:26:38 Cas9 Structural Dynamics and Genome Searching Kinetics
Protein Structure and Conformational Changes: The Cas9 protein structure changes significantly upon binding to guide RNA, creating a channel for DNA-RNA hybrid binding. A series of crystallographic structures illustrate the dramatic structural changes during Cas9 assembly with RNA and DNA. The Cas9 protein undergoes further conformational rearrangement to position its catalytic domain for DNA cleavage.
Kinetics and Mechanism of Genome Searching: Cas9-RNA complex rapidly binds and releases DNA, searching the genome through non-processive sliding. Binding to PAM sequences slows down complex release, allowing interrogation of adjacent DNA strands. Complementarity with the guide RNA initiates DNA unwinding and R-loop formation, leading to double-stranded break generation.
Cas9 Behavior in Living Cells: Super-resolution microscopy reveals the behavior of Cas9 in live cell nuclei. Apo Cas9 and Cas9 with non-binding guide RNAs exhibit rapid diffusion. Cas9 programmed with a guide RNA targeting multiple genomic sites shows slower diffusion and more frequent binding events.
00:32:36 Cas9 System Applications and Ethical Considerations
Cas9’s Behavior: Cas9 spends minimal time on off-target sites, rapidly releasing those with imperfect matches to the guide RNA. On-target sites with perfect matches result in Cas9 remaining bound for minutes or longer. Cas9 can navigate highly compacted heterochromatic regions of the genome, albeit with slower kinetics.
Challenges for Therapeutic Applications: Delivery: How to efficiently introduce Cas9 into specific cells or tissues for therapeutic editing. DNA Repair Control: Understanding and controlling the DNA repair process following Cas9-induced DNA cleavage. Ethical and Societal Implications: Considering the ethical and societal ramifications of using CRISPR for various applications.
Preassembled Protein RNA (RNP) Delivery: Preassembling Cas9 and guide RNA into RNP complexes can eliminate the need for introducing foreign nucleic acids into cells. RNP delivery allows rapid detection of editing within hours, minimizing potential off-targeting. Co-delivery of DNA templates enhances the rate of new DNA incorporation at the break site via homology-directed repair. Collaboration with colleagues to use RNP delivery to rewrite the sequence in primary human T cells for research and potential clinical applications.
Ethical Considerations and the Need for Discussion: With the advent of rapid and relatively easy genome editing, ethical and societal considerations must be addressed. Initial meeting in Napa Valley brought together scientists, clinicians, and stakeholders to discuss these issues.
00:38:32 Genome Editing Ethics and Safety Considerations
The Asilomar Meeting and the Prudent Path Forward: A small group of scientists, including Jennifer Doudna, David Baltimore, and Paul Berg, who had been involved in the Asilomar meeting in the 1970s around molecular cloning, met to discuss the ethical and safety considerations of using genome editing for clinical applications in human embryos. The outcome of this meeting was the publication of a paper in Science magazine in March 2015, proposing a “prudent path forward” by asking scientists to refrain from using genome editing for clinical applications in human embryos until further discussion and consideration of ethical, safety, and other relevant factors.
International Collaboration and Ongoing Discussions: The publication of the paper triggered global attention and discussion on the issue. A meeting was held in Washington sponsored by the Chinese Academy of Sciences, the Royal Society in the UK, and the National Academies of Science in the US to discuss these issues further. Additional meetings are planned for the year, and there will likely be increased media attention on the topic.
Jennifer Doudna’s Contributions and Collaborations: Doudna acknowledges her transition from being a RNA biochemist and structural biologist to exploring the applications of genome editing technology in various systems. She highlights the collaborative effort of the Innovative Genomics Initiative, involving UC Berkeley and UCSF, and encourages people to learn more about the initiative online. Doudna expresses gratitude for philanthropic donors and partnerships with companies to advance the application of genome editing technologies to specific disease targets. She acknowledges the contributions of her students, postdocs, and collaborators, especially Alex Marson, in helping to explore the clinical applications of genome editing.
Funding and Support: Doudna acknowledges the National Science Foundation (NSF) as a key funding source for her work, particularly for providing the first grant to work on CRISPR biology before widespread interest in the field. She expresses gratitude to all funding sources and attendees for their support.
Abstract
Revolutionizing Medicine and Ethics: The Impact and Implications of CRISPR-Cas9 Genome Editing
Precision Medicine: Overview and Challenges
Precision medicine aims to transition from traditional human judgment to evidence-based medicine and algorithms designed to improve average outcomes. The challenge lies in measuring and ensuring confidence in better outcomes for each patient. Achieving precision medicine requires a delicate balance between accuracy and practicality.
Pioneering Precision Medicine: The CRISPR-Cas9 Breakthrough
Jennifer Doudna, a Hilo native, made seminal contributions to precision medicine. Her journey from Hawaii to Harvard and the University of Colorado Boulder led to pioneering work in ribozymes and RNA interference pathways. Her collaboration with Jill Banfield unraveled the mysteries of the microbial immune system, CRISPR, culminating in the groundbreaking discovery of the CRISPR-Cas9 system.
Expanding the Horizons: Applications of CRISPR-Cas9
CRISPR-Cas9’s versatility extends to numerous applications. In research, it facilitates the study of gene functions and disease mechanisms. In medicine, it opens doors to gene therapies for genetic diseases and cancer. In agriculture, it promises improved crop yields and pest resistance. Its potential in industrial biotechnology cannot be understated, with applications ranging from biofuel production to other industrial processes.
Technical Mastery: Understanding the CRISPR-Cas9 System
The CRISPR-Cas9 system is characterized by its ease of use and adaptability, contributing to its wide application. The Cas9-guide RNA complex operates through a sequence of complex steps. These involve the integration of foreign DNA, RNA transcription and processing, and the formation of a ribonucleoprotein complex. This complex is vital for the rapid identification of target DNA sequences with high specificity. Cas9 demonstrates unique behavior in this context. It spends minimal time on off-target sites, quickly releasing those with imperfect matches to the guide RNA. In contrast, at on-target sites with perfect matches, Cas9 remains bound for extended periods. Furthermore, Cas9 can navigate the densely packed heterochromatic regions of the genome, although its kinetics are slower in these areas.
Navigating Ethical Waters: The Responsibility of Power
The power of CRISPR-Cas9 to edit the human genome introduces significant ethical and societal considerations. There are concerns about off-target effects, unintended consequences in complex organisms, and the moral implications of altering human traits. These considerations necessitate a cautious and responsible approach to using this technology, a stance advocated by Jennifer Doudna herself, particularly regarding human embryo editing. Challenges specific to therapeutic applications include developing efficient delivery methods to introduce Cas9 into target cells or tissues, understanding and controlling the DNA repair process post-Cas9-induced DNA cleavage, and considering the ethical and societal implications of various CRISPR applications. A promising development in this area is the preassembled Protein RNA (RNP) Delivery method. This approach involves assembling Cas9 and guide RNA into RNP complexes, eliminating the need to introduce foreign nucleic acids into cells. RNP delivery allows for rapid detection of editing, minimizing potential off-target effects, and enhances the rate of new DNA incorporation at the break site via homology-directed repair. This method is being explored in primary human T cells for research and potential clinical applications, as demonstrated in collaborations with colleagues.
Overcoming Challenges: The Path Forward in Therapeutics
Despite its revolutionary impact, CRISPR-Cas9 faces practical challenges in therapeutic applications. These include efficient delivery to specific cells or tissues, control over DNA repair processes, and addressing the broader ethical and societal implications. A promising approach in overcoming these challenges is the use of preassembled protein-RNA complexes (RNPs) for direct introduction into cells. This method has shown success in experiments involving human T cells.
A Call for Global Dialogue: Ethical and Societal Discourse
The advent of genome editing with CRISPR-Cas9 has sparked global discussions on its ethical, safety, and societal implications. Jennifer Doudna’s call for a cautious approach to human embryo editing, as published in Science magazine, is indicative of the scientific community’s awareness of these issues. This situation underscores the need for ongoing collaboration among researchers, clinicians, stakeholders, and the public in navigating the ethical landscape of genome editing. Ethical considerations and the need for discussion have become increasingly important with the rapid advancement of genome editing technology. The initial meeting in Napa Valley, which brought together scientists, clinicians, and stakeholders, highlighted these concerns. The Asilomar meeting, attended by prominent scientists including Jennifer Doudna, David Baltimore, and Paul Berg, focused on the ethical and safety considerations of using genome editing in clinical applications involving human embryos. This meeting led to the publication of a paper in Science magazine in March 2015, proposing a “prudent path forward” and urging scientists to refrain from using genome editing for clinical applications in human embryos until more thorough discussions and considerations of ethical, safety, and other relevant factors have taken place.
CRISPR: A Journey from Curiosity-Driven Research to Precision Medicine
The discovery of CRISPR-Cas9 was a result of curiosity-driven research. Jennifer Doudna, a Hilo girl with a passion for science, grew up in Hawaii, where she developed her curiosity about the natural world. The field of precision medicine seeks to understand the function of specific genetic information in relation to health and disease. In the late 1980s, scientists recognized the potential of making precise changes in DNA at the level of a specific gene or base pair to study gene function and develop new therapies.
Mechanism and Applications
CRISPR-based genome editing involves a protein called Cas9, guided by a short RNA sequence, which recognizes and cuts specific DNA sequences. By designing the RNA sequence, researchers can target specific genes and introduce precise changes, such as corrections to genetic defects or insertions of new genetic information. This technology has broad applications in basic research, biotechnology, and medicine, including the development of new therapies and the study of gene function. Jennifer Doudna has transitioned from being an RNA biochemist and structural biologist to exploring the applications of genome editing technology. She emphasizes the collaborative nature of the Innovative Genomics Initiative, involving UC Berkeley and UCSF. Doudna also expresses her gratitude for the support from philanthropic donors and partnerships with companies to advance genome editing technologies. She acknowledges the contributions of her students, postdocs, and collaborators, especially Alex Marson, in exploring the clinical applications of genome editing. The publication of the paper on genome editing triggered global attention and discussion. A notable meeting in Washington, sponsored by the Chinese Academy of Sciences, the Royal Society in the UK, and the National Academies of Science in the US, was held to further discuss these issues. Additional meetings are planned, likely attracting increased media attention.
Ethical and Societal Considerations
The development of powerful genome editing technologies raises ethical and societal questions, including concerns about potential misuse and unintended consequences. Jennifer Doudna emphasizes the importance of responsible and transparent approaches to the development and use of these technologies. Ongoing discussions and collaborations among scientists, ethicists, policymakers, and the public are crucial for addressing these complex issues. Doudna acknowledges the National Science Foundation (NSF) as a key funding source for her work, particularly for providing the first grant to work on CRISPR biology before widespread interest in the field. She expresses gratitude to all funding sources and attendees for their support.
CRISPR-Cas9 System: A Detailed Examination
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences are repetitive DNA segments found in bacteria and archaea. CRISPR-associated (Cas) genes are often located near CRISPR sequences and encode proteins involved in the CRISPR-Cas system. The CRISPR-Cas system acts as an adaptive immune defense mechanism in bacteria and archaea. It protects against foreign DNA, such as viral or plasmid DNA, by integrating foreign DNA sequences into the CRISPR locus. These integrated sequences are transcribed into RNA molecules and processed into smaller RNAs. These RNAs form complexes with Cas proteins to target and cleave matching DNA sequences, providing immunity against foreign DNA.
Cas9 is a key protein involved in the CRISPR-Cas system. It interacts with CRISPR RNA (crRNA) and tracer RNA (tracrRNA) to form a complex that binds to and cleaves DNA. The crRNA sequence determines the DNA sequence that will be targeted for cleavage. Martin Jinek simplified the CRISPR-Cas9 system by linking crRNA and tracrRNA into a single guide RNA (sgRNA) molecule. This sgRNA contains both the targeting information and the Cas9 binding information. The sgRNA can be easily altered to target different DNA sequences.
Conclusion
CRISPR-Cas9 stands as a beacon of scientific innovation, offering unprecedented possibilities in medicine, research, and beyond. Its journey is not just one of scientific discovery but also of ethical contemplation. As we forge ahead, the balanced interplay of scientific advancement and responsible stewardship will be crucial in harnessing the full potential of this extraordinary technology.
CRISPR technology has revolutionized genetic engineering, enabling precise DNA modifications with potential for treating genetic diseases and developing novel therapies, but ethical and regulatory considerations are essential. CRISPR's versatility extends beyond DNA editing, with applications in RNA targeting and genome editing in various organisms, raising ethical questions about its use...
The CRISPR-Cas9 system, pioneered by Jennifer Doudna, revolutionized genome editing with its ability to target specific DNA sequences, opening up new avenues in biomedical research and gene therapy. CRISPR-Cas9's dual RNA-guided DNA cleaving mechanism enables precise genome editing and has profound implications in the fields of medicine and genetics....
CRISPR-Cas9 technology revolutionized gene editing with its precise modifications of DNA, but raises ethical questions regarding its use and potential consequences. Scientists, audiences, and regulators must engage in responsible discussions to guide ethical development and application of CRISPR....
CRISPR-Cas9, pioneered by Jennifer Doudna, revolutionized gene editing, enabling precise DNA manipulation with implications for medicine, agriculture, and research. The technology's ethical considerations, such as germline editing and designer babies, necessitate responsible progress and discussion....
CRISPR-Cas9, a revolutionary gene-editing technology, allows precise editing of DNA, with applications in treating genetic diseases, enhancing agricultural practices, and potentially improving human traits. CRISPR-Cas9 can be used to make targeted changes in the microbiome, potentially benefiting human health and reducing environmental impact....
CRISPR, a revolutionary gene-editing technology, has profound ethical, scientific, and regulatory implications, particularly in the context of human genome editing. The ease of access and accessibility of CRISPR raise concerns about potential misuse and the need for balancing scientific progress with responsible use....
CRISPR-Cas9 is a revolutionary gene-editing tool that uses RNA molecules to target and cleave specific DNA sequences. The system's discovery has revolutionized genome editing, offering a powerful tool for precise genetic modifications....