Historical Context:
Alan S. Michaels, an MIT chemical engineering pioneer, established the Alan S. Michaels Lectureship in Medical and Biological Engineering in 1995. Dr. Michaels’ research focused on colloid phenomena, soil mechanics, and polymer permeability, leading to the development of perm-selective polymeric membranes and the founding of Amicon Corporation.

Dr. Jennifer Doudna: A Renowned Expert in CRISPR-Cas9 Technology
Dr. Jennifer Doudna, a renowned professor of chemistry, molecular and cell biology at UC Berkeley, revolutionized the field of gene editing with her groundbreaking work on CRISPR-Cas9. Dr. Doudna and her colleagues developed a simple method for editing DNA using RNA-guided proteins, opening up vast possibilities for research and applications in human and non-human gene editing.

CRISPR-Cas9: An Adaptive Immune System in Bacteria
CRISPR-Cas9 is a natural defense mechanism found in bacteria that protects them from viruses. It consists of a locus of repetitive DNA elements interspersed with unique sequences derived from viruses, forming a genetic record of previous viral exposures. CRISPR-associated (Cas) proteins produce transient RNA copies of the viral sequences, which combine with Cas proteins to form complexes that identify and cut matching DNA sequences, selectively destroying viral DNA without harming the bacterial genome.

Dr. Doudna’s Fascination with RNA and CRISPR-Cas9
Dr. Doudna’s background in RNA biology and structural biology drew her to the CRISPR-Cas9 system. She recognized the immense potential of this adaptive immune system for gene editing and embarked on research to understand its molecular mechanisms and explore its applications.

CRISPR-Cas9 Applications and Future Considerations
CRISPR-Cas9 has revolutionized gene editing, enabling precise modifications to DNA in a wide range of organisms, including humans. It holds promise for treating genetic diseases, developing new therapies, and advancing agricultural research. However, the widespread use of CRISPR-Cas9 raises ethical and societal questions regarding its potential impact on human health, the environment, and the implications of altering the genetic makeup of living organisms.

Unraveling the Secrets of Bacterial Immunity:
Jennifer Doudna’s curiosity was sparked by Jill Banfield’s discovery of RNA-guided systems in bacteria, challenging the notion that such mechanisms were exclusive to plants and animals. Initial research focused on understanding the function of proteins and RNAs involved in adaptive immune systems, leading to a collaboration with Emmanuel Charpentier’s lab.

The Role of Cas9 in DNA Cleavage:
The aim was to decipher the function of Cas9, a protein implicated in RNA-guided protection against infection in bacteria. Cas9 employs an RNA guide to interact with specific DNA sequences and triggers double-stranded DNA cleavage, precisely controlled by the RNA guide sequence.

Dissection of the CRISPR-Cas9 System:
Martin Jinek and Chris Chylinski collaborated to investigate the molecular components of the CRISPR-Cas9 system. They discovered that Cas9 naturally uses two distinct RNAs, CRISPR RNA and tracer RNA, to form a functional complex for guided DNA cleavage.

Simplifying the System:
Martin Jinek’s crucial experiment involved linking the two separate RNAs into a single guide RNA, maintaining the ability to bind to Cas9 and introducing a 20-nucleotide sequence for precise DNA targeting. This breakthrough transformed the project from a basic immunity investigation to a potentially groundbreaking tool for genome editing.

Implications in Cell Types Beyond Bacteria:
Double-stranded breaks in bacteria typically lead to DNA destruction, serving as an effective immune mechanism. In contrast, animal and plant cells respond differently to double-stranded breaks, enabling DNA repair and sequence alteration at the break site.

Advantages of Cas9 over Existing Technologies:
Cas9, unlike earlier technologies like zinc finger nucleases and talons, is a single protein that can be effortlessly reprogrammed using its RNA guide. The programmability of Cas9, coupled with the understanding of its targeting mechanism, eröffnet enormous potential for genetic sequence manipulation.

The CRISPR Revolution:
Publication of the CRISPR-Cas9 genome editing system in 2012 sparked a surge of interest and applications worldwide. The technology’s potential for genome editing and manipulation raised important ethical questions and the need for careful consideration of its implications.

Focus on CRISPR Genome Editing Toolbox and Ongoing Research:
Doudna’s current research delves into the CRISPR genome editing toolbox, exploring its mechanisms and potential improvements. The goal is to advance the technology for future applications in various fields.

Two Families of CRISPR-Cas Enzymes:
Currently, two families of CRISPR-Cas enzymes are utilized for genome editing: Class 1 and Class 2. These enzymes offer versatile applications, including genome editing, transcriptional control, and imaging experiments.

Type 2 and Type 5 CRISPR-Cas Enzymes:
Two classes of CRISPR-Cas enzymes exist: Type 2 (Cas9 type proteins) and Type 5 (Cas12, CasX, and other emerging enzymes). Type 5 enzymes display greater natural diversity compared to Type 2 enzymes.

Mechanism of Type 2 Enzymes:
Cas9 proteins possess two active sites that cut DNA, generating blunt cuts. They utilize a dual RNA guide, combining two separate RNAs into a single guide type RNA.

Mechanism of Type 5 Enzymes:
Type 5 enzymes have a single active site for DNA cleavage, enabling them to cut both strands of the DNA double helix. They generate staggered breaks in DNA and naturally employ a single guide RNA for address labeling.

Key Differences:
Type 2 enzymes have two active sites, make blunt cuts, and use a dual RNA guide. Type 5 enzymes have a single active site, make staggered cuts, and use a single guide RNA.

Focus of the Presentation:
The presentation will delve into the mechanisms of Type 5 CRISPR-Cas systems, exploring their unique features and potential applications. It will also discuss how nature controls these enzymes, the identification of control proteins, and their potential use in future research.

Cas12 Proteins Possess an Additional Chemical Activity:
Cas12 proteins exhibit an unexpected ability to cut single-stranded DNA molecules non-specifically after recognizing a target site through RNA-guided recognition. This activity may be attributed to the geometry of the enzyme and the dynamic nature of its single active site, allowing it to move around and cut both strands of a double-stranded substrate.

Cas12’s Single Active Site and Its Sequential Cleavage Mechanism:
Cas12’s single active site enables it to cut both strands of a DNA double helix in a sequential manner. Initial cleavage occurs on the non-target strand, followed by trimming back of the nucleotides. This trimming activity is crucial for the enzyme’s ability to ultimately make a double-stranded DNA break.

Implications for Nickase Versions of Cas12-Type Proteins:
The sequential cutting mechanism of Cas12 proteins poses challenges in creating nickase versions of these enzymes. Introducing a single break in the DNA backbone is insufficient to achieve double-stranded cutting; trimming away of nucleotides is necessary. Mutations aimed at creating nickase versions of Cas12 proteins result in extremely slow cutting rates, hindering their practical application.

Cas12 Type Proteins Diversity:
Type five Cas12 proteins exhibit notable diversity in nature.

Collaboration with Jill Banfield’s Lab:
Joint research with the Banfield Lab focused on analyzing CRISPR systems across diverse microbes to identify new classes of enzymes with potentially interesting functions.

CRISPR System Analysis:
A study revealed three distinct types of proteins, including an archaeal Cas9, which expanded the understanding of Cas9’s presence beyond bacteria. Two novel CRISPR-Cas enzymes, CasX and CasY, were identified, primarily originating from uncultivated microbes.

CasX Protein Characteristics:
CasX belongs to the type five CRISPR-Cas enzyme group. Similar to Cas12, CasX possesses a single active site for DNA cutting.

Metagenomic DNA Analysis:
The Banfield Lab employs metagenomic DNA analysis to assemble genomes and identify CRISPR arrays flanked by CRISPR-Cas enzyme genes. The adaptive step of the immune system involves integrase proteins responsible for capturing viral DNA and integrating it into the CRISPR sequence.

CasX Gene Identification:
CasX was identified due to its unique genetic sequence, lacking homology to other CRISPR-Cas enzymes, except for the RUBC domain, a cleavage domain commonly found in CRISPR-Cas proteins like Cas9 and Cas12.

Origins of CasX Discovery:
CasX was initially discovered in a computational analysis of microbial genomes by David Burstein in the Banfield lab. Lucas Harrington, a bioengineering student, joined Burstein to experimentally investigate CasX.

Experimental Approach to Determine CasX Function:
Burstein and Harrington engineered the CasX locus to be expressed in E. coli. They tested the ability of CasX to recognize a plasmid DNA molecule containing a selectable marker.

Results of CasX Experiments:
Transformation assays showed that CasX is an RNA-guided DNA targeting enzyme. CasX specifically targets plasmid DNA with a matching targeting sequence. Mismatches between the targeting sequence and plasmid DNA disrupt CasX activity.

Structural Biology of CasX:
Junjie Liu and Natalia Orlova used cryo-electron microscopy and crystallography to study the structure of CasX. CasX is a complex of protein and RNA, with the RNA serving as the dominant scaffold. CasX undergoes conformational changes upon binding to DNA, leading to DNA cleavage.

Comparison of CasX with Cas9 and Cas12:
CasX shares similarities with Cas9 and Cas12 in terms of its RNA-guided DNA targeting mechanism. However, CasX has a unique structure and mechanism of action compared to these other CRISPR-Cas systems.

Potential Applications of CasX:
CasX has potential applications in genome editing, diagnostics, and other biotechnology fields.

Structural Insights into CasX:
CasX structure: Large protein with RNA scaffold and active site for DNA cleavage. Initial state: DNA paired with guide RNA opened in enzyme, active site poised for first cut. Second conformation: Target DNA strand drops into active site, allowing second cleavage. Structural mapping of cutting positions reveals 10 base pair overhang, consistent with biochemical observations.

Sequential DNA Cleavage Mechanism:
CasX exhibits sequential cutting of DNA strands: first strand cleavage followed by trimming. Second cleavage event occurs once RNA-DNA helix drops into the enzyme active site. Structural analysis supports the observed 10 base pair overhang generated during DNA cleavage.

Control of CRISPR-Cas Enzymes:
Nature’s defense against CRISPR-Cas: Anti-CRISPR proteins that inhibit enzyme activity. Anti-CRISPRs: Small proteins with diverse mechanisms to block CRISPR-Cas function.

Discovery of Anti-CRISPRs:
Initial studies: Identification of phages not cleared by CRISPR-Cas systems and small proteins near CRISPR arrays. Kyle Waters’ approach: Automating the search for anti-CRISPRs through analysis of CRISPR array sequences. Self-targeting sequences: Bacterial genomes with CRISPR systems containing sequences recognizing sites within their own genome. Self-targeting sequences often correspond to integrated phage sequences (prophage).

Genome Engineering with CRISPR-Cas Systems:
Jennifer Doudna discusses the potential of CRISPR-Cas systems for genome engineering and the need to identify inhibitors to control their activity.

Self-Targeting Sequences and CRISPR Inhibitors:
Kyle Waters’ research revealed a significant number of self-targeting sequences associated with different CRISPR systems, suggesting the widespread presence of CRISPR inhibitors across the microbial world.

Inhibitor Discovery Strategy:
Kyle Waters devised an assay to screen for CRISPR inhibitors using transcription translation extracts, reporter plasmids, Cas12-containing CRISPR systems, and genome fragments containing self-targeting sequences.

Identification of Cas12 Inhibitors:
Through this screening process, three unique inhibitors of Cas12 were identified, including one called anti-CRISPR 5A1 (5A1).

Mechanism of Action of 5A1:
Gavin Knott’s experiments demonstrated that 5A1 is a robust inhibitor of Cas12a, working at low ratios of inhibitor to enzyme and suggesting a multiple turnover mechanism.

5A1 as an RNase:
Surprisingly, 5A1 was found to be an RNase that cleaves the guide RNA for Cas12 at a specific position adjacent to the DNA-recognition sequence.

Implications for CRISPR-Cas Research and Applications:
The discovery of CRISPR inhibitors like 5A1 provides valuable tools for controlling CRISPR-Cas activity, enhancing their safety and specificity for genome editing and other applications.

Inhibitor Mechanism:
The inhibitor leaves the handle part of the guide intact, making it difficult for Cas12 to bind and reload a fresh guide. The inhibitor binds to a specific place on Cas12 to become activated and cut the guide RNA. Active site residues in the inhibitor, such as conserved histidines, are essential for its function. The inhibitor binds, cuts off the guide, falls off, and can then bind to a new Cas12 and repeat the process, resulting in high turnover rates.

Future Prospects of CRISPR-Cas:
Mechanistic studies can uncover unexpected activities of CRISPR-Cas proteins. The target-activated DNA cleavage ability of Cas12 is being developed as a point-of-care diagnostic tool to detect DNA molecules. CRISPR-Cas systems have potential applications in agriculture, such as increasing crop yield and disease resistance. CRISPR-Cas holds promise in the development of new treatments for genetic diseases and cancer.

Key Points:
New Opportunities for CRISPR: CRISPR-CasX, a new type of CRISPR enzyme, has a different ratio of RNA to protein, opening possibilities for engineering and delivery in low-tech settings. Ethical Implications: As genome editing advances, ethical considerations arise. Germline editing in humans is a particular concern due to potential unintended consequences. The He Jiankui Case: He Jiankui’s announcement of germline edits in twin girls raised concerns about the safety and readiness of the technology. Global Guidelines Needed: International forums are being convened to establish clear and restrictive guidelines for germline editing in humans. Balanced Approach: Balancing the responsible use of the technology with ensuring its accessibility for research and clinical applications is crucial.

Delivery and Control in RNA-Guided Gene Regulation:
Delivery Methods: CRISPR-Cas systems face challenges in delivery to target cells, especially in complex organisms like humans. Researchers are exploring various methods, including cell-penetrating peptides, nanoparticles, and viral vectors. Chemical Control: Control over CRISPR activity is vital to prevent unintended consequences. Chemical modifications to the CRISPR components, such as guide RNA and Cas proteins, can fine-tune their activity and minimize off-target effects.

Societal Control and Guidelines:
Societal Oversight: The rapid advancement of gene editing technologies has prompted calls for societal oversight and guidelines. International forums are being convened to establish clear criteria for germline editing in humans, considering ethical, legal, and societal implications. Responsible Use: The scientific community has a responsibility to ensure the responsible use of gene editing technology. This includes adhering to ethical guidelines, promoting transparency, and advocating for public engagement in decision-making.

Fundamental Research Drives Technology Development:
Understanding Fundamental Systems: Fundamental research continues to drive the development of CRISPR technology. Studying the mechanisms and structures of CRISPR-Cas systems in bacteria provides insights for harnessing and controlling them for various applications. Collaboration and Partnerships: Collaborative efforts among researchers, clinicians, and ethicists are essential for responsible and effective development of gene editing technologies.

RNA-Guided Adaptive Immunity:
Adaptive Immunity in Bacteria: Bacteria possess RNA-guided adaptive immunity systems that allow them to acquire sequences from viruses or foreign DNA. This enables them to adapt to infections over multiple generations.

CAS-X Properties and Therapeutic Potential:
CAS-X Properties: CAS-X has properties intermediate between CAS-9 and CAS-12, offering potential advantages for therapeutic applications. RNA Editing: The RNA editing capabilities of CAS-X are being investigated due to its single nuclease activity. Immunogenicity: The immunogenicity of CAS-X is being studied, considering its origin from a non-pathogenic bacteria. This could impact its potential use as a therapeutic.

CRISPR-Cas12 Immunogenicity and CasX:
Cas12, like Cas9, may face challenges due to pre-existing antibodies in humans since it originates from a pathogenic bacterium. CasX, derived from soil bacteria, lacks pre-existing antibodies and thus reduces the risk of immune reactions. CasX specifically targets double-stranded DNA and does not interact with RNA.

Evolution of CRISPR Systems:
The selective pressure from viruses is a primary factor driving the evolution of CRISPR systems, as they provide antiviral protection. Many acquired sequences in CRISPR arrays remain unmapped, suggesting potential targeting of unidentified viruses or other functions. Recent findings indicate that CRISPR arrays can target the genomes of neighboring bacteria in symbiotic biofilms, hinting at additional regulatory roles.

Kinetics of Inhibition and DNA Cutting:
The inhibitor’s ability to bind and cut the guide versus the enzyme’s rate of assembly and DNA cutting is being investigated. Understanding the kinetics of inhibition and DNA cutting will shed light on the efficiency and specificity of CRISPR-based gene editing.

Genome-Line Editing and Ethical Considerations:
Human embryo editing is currently feasible, but accuracy and stability remain challenges. Advances in DNA repair mechanisms and functional genomics will improve the precision of embryo editing in the future. Balancing the potential benefits and risks of germline editing is crucial, with careful consideration of medically necessary and desirable uses. The field needs ongoing vigilance and ethical discussions to determine when and how to responsibly utilize embryo editing technology.

Additional Cas Activities:
Cas proteins can target single-stranded RNA, displaying additional activity in cutting single-stranded RNA. The function of these proteins in their native bacterial hosts remains unknown. Single-stranded targeting activity might be useful for targeting single-stranded DNA phages or RNA phages. Challenges in studying these proteins include difficulties in culturing the bacteria and identifying their phages.

Ethical and Societal Implications:
Jennifer Doudna emphasizes the importance of considering the ethical and societal implications of CRISPR-Cas systems. She acknowledges the need for scientists to embrace these questions. Doudna suggests that scientists should have nerves of steel to navigate these complex issues.

Addressing the Need for Training:
Current graduate educational programs often lack systematic training in addressing ethical and societal implications. Doudna emphasizes the need for programmatic training to equip students with the necessary skills. Universities and biomedical programs should consider incorporating such training into their curricula.

Learning and Adapting:
Doudna emphasized the importance of continuous learning and seeking knowledge outside her field of expertise. She mentioned how she had to learn about IVF and human embryogenesis to understand the ethical implications of gene editing.

Understanding the Law:
Doudna stressed the need for scientists to understand the laws and regulations governing their research, as they vary across different countries.

Accessible Explanations:
Doudna highlighted the challenge of explaining scientific work in a way that is accessible to non-experts. She suggested that scientists develop a few simple sentences to summarize their work and its significance to laypeople.

Breaking Down Barriers:
Doudna expressed concern about the suspicion and mistrust of science and scientists in society. She believes that better communication of the scientific process, including the iterative nature of research, could help bridge this gap.

Improving Connections:
Doudna expressed hope that improved communication between scientists and the public could foster better connections and understanding across society.