Jennifer Doudna (UC Berkeley) (Dec 2015)

Jennifer Doudna (UC Berkeley Professor) – Breakthrough Prize Symposium (Dec 2015)

Chapters

00:00:10 CRISPR-Cas9 Technology: A New Era of Precision Genome Editing

Introduction:
CRISPR-Cas9 technology emerged from a curiosity-driven research project in Jennifer Doudna’s lab at Berkeley. The project aimed to investigate how bacteria fight viral infections.

Discovery of CRISPR Sequences:
Jillian Banfield, a colleague of Doudna, observed unusual sequence signatures, called CRISPRs, in bacterial genomes. CRISPRs consist of repeated DNA sequences interspersed with unique sequences. These unique sequences often matched sequences found in viruses that infect bacteria.

CRISPR as a Bacterial Immune System:
Jill Banfield hypothesized that CRISPRs might play a role in a bacterial immune system. Bacteria may record viral infections, store the genetic information in CRISPR sequences, and use it to protect against future infections. RNA molecules, derived from CRISPR sequences, may be involved in this process.

Experimental Evidence for CRISPR Immunity:
Researchers at Danisco showed that bacteria with CRISPR loci can acquire immunity to phage infections. Foreign DNA from viruses is integrated into the CRISPR locus, allowing bacteria to recognize and cut up viral DNA.

Mechanism of CRISPR-Cas9 Targeting:
CRISPR-Cas9 technology involves a protein called Cas9 and RNA molecules. Cas9 is programmable with CRISPR-derived RNA molecules. The RNA molecules guide Cas9 to specific DNA sequences for targeted double-stranded DNA cutting.

Development of a Single-Component Guide RNA:
Martin Jinek simplified the CRISPR-Cas9 system by creating a chimeric single guide RNA. This RNA has both targeting and Cas9 binding capabilities, enabling precise DNA cutting.

CRISPR-Cas9 in Animal and Plant Cells:
Cells have sophisticated machinery to repair double-stranded DNA breaks. CRISPR-Cas9 can induce double-stranded DNA breaks at specific sites in genomes. These breaks can be repaired through non-homologous end joining or homologous recombination, introducing genetic changes.

Conclusion:
CRISPR-Cas9 technology allows for precise editing of DNA in cells, providing a powerful tool for genetic research and therapeutic applications.

00:11:07 CRISPR Technology: A Platform for RNA-Programmed Genome Editing

Background:
Traditional genome editing techniques were labor-intensive, expensive, and required specialized expertise. Jennifer Doudna and her colleagues aimed to create a versatile platform for genome editing using a single protein and short guide RNAs.

CRISPR-Cas9 Technology:
CRISPR-Cas9 utilizes a protein (Cas9) that can be reprogrammed with a short guide RNA to create targeted double-stranded breaks in DNA. This RNA-programmed genome editing approach is adaptable and can be easily tested in various systems.

Rapid Adoption of CRISPR-Cas9:
The CRISPR-Cas9 technology has seen exponential adoption worldwide due to its simplicity and effectiveness. It allows for genome engineering in various organisms, including human cells and zebrafish, making it a versatile platform.

Advantages of CRISPR-Cas9:
It is analogous to software, allowing reprogramming with small RNA fragments, making it easily adaptable to different systems. It works in various animal and plant systems, offering precision in editing. Its mechanism involves rapid and accurate DNA recognition, crucial for applications requiring precision.

Applications of CRISPR-Cas9:
CRISPR-Cas9 has been employed for engineering plants, animals, and organisms previously considered genetically intractable. It enables targeted gene editing, gene activation/deactivation, and genome imaging. The multiplexing capability allows simultaneous editing at multiple genomic locations.

Understanding the Mechanism:
Jennifer Doudna’s lab focuses on understanding the molecular mechanism of CRISPR-Cas9 to ensure its safe and effective use in curing genetic diseases.

Modified Cas9 Variants:
Researchers have modified Cas9 to create a deactivated version for DNA binding without cleavage, enabling gene regulation and imaging applications. This approach doesn’t involve genome engineering or double-stranded breaks.

Natural Multiplexing Capability:
CRISPR-Cas9 can be programmed with multiple guide RNAs, allowing simultaneous editing at different genomic locations in a single experiment. This capability enhances experimental efficiency and enables comprehensive genetic analysis.

Rapid and Accurate DNA Recognition:
CRISPR-Cas9’s ability to rapidly and accurately recognize DNA targets is crucial for its precision and versatility. Recent data obtained by Doudna’s lab provides insights into the mechanisms underlying this recognition process.

00:17:26 How Cas9 Protein Undergoes Conformational Changes

00:21:20 Cas9 Genome Editing: Challenges and Prospects for Therapeutic Applications

Nucleosome Structure and Cas9 Binding:
Bacterial genomes are smaller and lack chromatin structures compared to eukaryotic genomes. Cas9 protein deals with compacted DNA structures found in eukaryotic cells, but questions arise regarding its interaction with chromatin and larger mammalian genomes.

Cas9 Particle Visualization:
Cas9 complexes are labeled with fluorescent markers to visualize their movement within live cell nuclei. Rapid movement of particles is observed with a nonsense guide RNA, indicating non-specific binding. Targeting a repetitive element in the mammalian genome results in slower particle movement, suggesting binding to specific sites.

Measuring Kinetics and Target Search:
Experiments measure the kinetics of target search by Cas9 in the cell. Ongoing research aims to determine the time taken for a single Cas9 particle to find a target site in the human genome. Understanding these kinetics is essential for efficient editing and minimizing off-target effects.

Challenges in CRISPR Technology:
Delivery of CRISPR components into tissues remains a major challenge. Controlling the way DNA is repaired after the CRISPR cut is another hurdle.

RNP Delivery and Advantages:
Preassembled protein RNA complexes (RNPs) are used for CRISPR delivery, enabling rapid editing within hours. The half-life of the RNP complex minimizes off-target effects. Co-delivery of DNA templates for repair improves control over the repair pathway.

Editing Primary Human T-cells:
CRISPR-mediated editing of human T-cells has been successful, including gene knockouts and knock-ins. This approach opens up possibilities for targeted editing of immune cells for cancer immunotherapy and research.

Ethical Considerations and Global Conversation:
The potential for editing the human germline raises ethical and societal questions. A call for a global conversation involves scientists and stakeholders to discuss safe and appropriate use of CRISPR technology.

Conclusion:
CRISPR technology has the potential for impactful clinical applications. Funding and collaboration are essential for advancing research and addressing challenges. Ethical considerations are paramount when considering germline editing.

00:31:48 Off-Target Activity and Cleavage Mechanism of Cas9

Abstract

CRISPR-Cas9: Revolutionizing Genome Editing and Beyond

Abstract

Jennifer Doudna’s groundbreaking journey in developing CRISPR-Cas9, a transformative gene-editing technology, is a tale of scientific ingenuity and interdisciplinary collaboration. Originating from a simple question about DNA alteration, this adventure uncovered the mechanisms of bacterial immunity, leading to the discovery of a tool that can precisely edit the genome. CRISPR-Cas9, derived from a bacterial defense system against viruses, consists of the Cas9 protein and guide RNA, collaborating to induce targeted DNA breaks. This technology’s applications range from treating genetic diseases to improving agricultural yields. Despite its vast potential, CRISPR-Cas9 raises significant ethical and societal concerns, necessitating a global dialogue for responsible usage.

Introduction

Jennifer Doudna’s foray into the world of CRISPR-Cas9 began with a curiosity-driven inquiry into the possibility of altering DNA sequences with precision. This exploration, fueled by a collaboration with Jillian Banfield, led to the discovery of a bacterial immune system characterized by CRISPRs. Doudna hypothesized an RNA-mediated defense mechanism, later confirmed through collaborative research. The pivotal discovery of Cas9’s role in DNA cleavage and the subsequent simplification of the system by Martin Jinek laid the foundation for CRISPR-Cas9 as a potent genome editing tool.

The CRISPR-Cas9 Mechanism and Its Applications

CRISPR-Cas9 operates by guiding the Cas9 protein to specific DNA sequences, creating double-stranded breaks. This system’s simplicity and versatility make it superior to previous technologies. Applications extend across various fields, including medicine for treating genetic disorders, agriculture for enhancing crop resilience, and even renewable energy development. Nevertheless, CRISPR-Cas9’s power brings ethical challenges, including concerns about unintended genome editing consequences and potential misuse.

*CRISPR-Cas9 technology involves a protein called Cas9 and RNA molecules. Cas9 is programmable with CRISPR-derived RNA molecules. The RNA molecules guide Cas9 to specific DNA sequences for targeted double-stranded DNA cutting.*

*Bacterial genomes are smaller and lack chromatin structures compared to eukaryotic genomes. Cas9 protein deals with compacted DNA structures found in eukaryotic cells, but questions arise regarding its interaction with chromatin and larger mammalian genomes.*

*CRISPR-Cas9 can be programmed with multiple guide RNAs, allowing simultaneous editing at different genomic locations in a single experiment. This capability enhances experimental efficiency and enables comprehensive genetic analysis.*

*CRISPR-Cas9’s ability to rapidly and accurately recognize DNA targets is crucial for its precision and versatility. Recent data obtained by Doudna’s lab provides insights into the mechanisms underlying this recognition process.*

Protein Structure and Kinetics in CRISPR-Cas9 Function

The Cas9 protein undergoes significant conformational changes during its interaction with nucleic acids, vital for its function. Fluorescence studies have elucidated these changes, revealing the active state of Cas9 upon DNA binding. Understanding these dynamics, both in vitro and in vivo, is crucial for comprehending and improving CRISPR-Cas9’s efficiency and specificity.

*CRISPR-Cas9 technology has seen exponential adoption worldwide due to its simplicity and effectiveness. It has been employed for engineering plants, animals, and organisms previously considered genetically intractable. It enables targeted gene editing, gene activation/deactivation, and genome imaging. The multiplexing capability allows simultaneous editing at multiple genomic locations.*

*The Cas9 protein undergoes significant structural rearrangement when it assembles with nucleic acids, transitioning from a closed to an open conformation. The protein’s two structural parts move apart to form a channel in the center, accommodating the DNA ends once the complex assembles with a target DNA molecule.*

*High-resolution crystallographic structures of Cas9 in different states reveal a remarkable structural change upon nucleic acid assembly. The protein undergoes conformational changes to accommodate RNA and DNA substrate binding.*

*Cas9 complexes are labeled with fluorescent markers to visualize their movement within live cell nuclei. Rapid movement of particles is observed with a nonsense guide RNA, indicating non-specific binding. Targeting a repetitive element in the mammalian genome results in slower particle movement, suggesting binding to specific sites.*

*A crucial domain for DNA cutting, the catalytic cleaver, is initially positioned incorrectly in the protein. It needs to swing into place to become a functional cleaver and cut the DNA.*

*Experiments measure the kinetics of target search by Cas9 in the cell. Ongoing research aims to determine the time taken for a single Cas9 particle to find a target site in the human genome. Understanding these kinetics is essential for efficient editing and minimizing off-target effects.*

Advancements in Delivery and Precision

Recent developments focus on delivering Cas9 more effectively, such as through preassembled RNPs, and enhancing genome editing precision. These advancements have facilitated rapid and precise genome alterations while reducing off-target effects. Particularly notable is the application in T-cell editing, presenting promising avenues in cancer immunotherapy.

*CRISPR-Cas9 technology has seen exponential adoption worldwide due to its simplicity and effectiveness. It has been employed for engineering plants, animals, and organisms previously considered genetically intractable. It enables targeted gene editing, gene activation/deactivation, and genome imaging. The multiplexing capability allows simultaneous editing at multiple genomic locations.*

*Researchers have modified Cas9 to create a deactivated version for DNA binding without cleavage, enabling gene regulation and imaging applications. This approach doesn’t involve genome engineering or double-stranded breaks.*

*Preassembled protein RNA complexes (RNPs) are used for CRISPR delivery, enabling rapid editing within hours. The half-life of the RNP complex minimizes off-target effects. Co-delivery of DNA templates for repair improves control over the repair pathway.*

*Editing Primary Human T-cells: CRISPR-mediated editing of human T-cells has been successful, including gene knockouts and knock-ins. This approach opens up possibilities for targeted editing of immune cells for cancer immunotherapy and research.*

Ethical Considerations and Future Outlook

The potential of germline editing and the societal implications of CRISPR-Cas9 necessitate a comprehensive ethical framework. The global scientific community must engage in an ongoing conversation to develop guidelines ensuring CRISPR-Cas9’s responsible and beneficial use. As research progresses, addressing delivery challenges, controlling DNA repair, and reducing off-target effects remain critical areas.

*The potential of germline editing and the societal implications of CRISPR-Cas9 necessitate a comprehensive ethical framework. The global scientific community must engage in an ongoing conversation to develop guidelines ensuring CRISPR-Cas9’s responsible and beneficial use.*

*CRISPR-Cas9 can be programmed with multiple guide RNAs, allowing simultaneous editing at different genomic locations in a single experiment. This capability enhances experimental efficiency and enables comprehensive genetic analysis.*

*CRISPR-Cas9’s ability to rapidly and accurately recognize DNA targets is crucial for its precision and versatility. Recent data obtained by Doudna’s lab provides insights into the mechanisms underlying this recognition process.*

Conclusion

CRISPR-Cas9 represents a significant leap in genome editing capabilities. Its development, stemming from a blend of curiosity and scientific collaboration, has opened doors to myriad applications. While the excitement around its potential is well-founded, the ethical and societal considerations it brings cannot be overlooked. The future of CRISPR-Cas9 lies in harnessing its power responsibly, ensuring its benefits are realized while mitigating its risks.

Notes by: Ain

Jennifer Doudna (UC Berkeley Professor) – Breakthrough Prize Symposium (Dec 2015)

Chapters

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