Jennifer Doudna (UC Berkeley Professor) – Microbiology Society Price – Accepted by Dr. Fellmann (Jun 2019)
Chapters
00:00:15 CRISPR-Cas: A Revolutionary Tool for Molecular Biology and Precision Medicine
Introduction to CRISPR-Cas: CRISPR-Cas is a ground-breaking tool in molecular biology and precision medicine that has transformed our approach to biomedicine and various biological studies. It allows for precise manipulation of genetic information, enabling us to study disease models, investigate single nucleotide polymorphisms, conduct drug screenings, assess safety and toxicology, develop cellular therapies, and explore potential therapeutics for various diseases.
Origins of CRISPR-Cas: The development of CRISPR-Cas began with a curiosity about how bacteria defend themselves against viral infections. Traditional molecular biology assumes a unidirectional flow of genetic information from DNA to mRNA to protein, and most current therapeutics target proteins. However, the question arose: what if we could modify the genetic code itself to address the underlying cause of diseases?
Discovery and Development of CRISPR-Cas: The discovery and advancement of CRISPR-Cas as a genome engineering tool by Jennifer Doudna and numerous collaborators worldwide has revolutionized our understanding and approach to biology, biomedicine, and agricultural technologies. This adaptive immune system from bacteria has been adapted as a powerful tool for biomedicine, recognized by the Dutton Prize awarded to Jennifer Dutton.
Benefits of CRISPR-Cas: CRISPR-Cas offers several advantages, including: High precision and specificity in gene editing. Versatility in targeting a wide range of genes and genomic regions. Applicability in various organisms, including humans, animals, plants, and microbes. Potential for developing novel therapies and treatments for genetic diseases and disorders.
Introduction to CRISPR-Cas Immune System: CRISPR-Cas is an adaptive immune system in bacteria that protects against bacteriophages through a three-step mechanism: adaptation, CRISPR biogenesis, and interference. Cas9, a key Cas protein, is guided by a single guide RNA to target specific DNA sequences and induce double-strand DNA cleavage. Double-strand DNA breaks can be repaired via non-homologous end joining (NHEJ) or homologous recombination (HR), enabling gene knockout or precise genome editing.
Natural Anti-CRISPR Proteins as Control Mechanisms: Anti-CRISPR proteins are natural phage-derived molecules that can regulate CRISPR-Cas activity. ACR5A1, 5A4, and 5A5 are anti-CRISPR proteins targeting Cas12a. Identification of anti-CRISPRs was achieved through computational analysis of self-targeting genomes and subsequent in vitro validation. Anti-CRISPRs protect Cas12a proteins from different bacterial origins, providing a versatile tool for controlling CRISPR-Cas activity.
Leveraging Anti-CRISPRs in Mammalian Cells: Anti-CRISPRs can inhibit Cas12a activity in mammalian cells. HEK-RT1 reporter cell line was developed to study Cas12a activity and anti-CRISPR efficacy. Anti-CRISPRs stably expressed in HEK-RT1 cells provide protection against Cas12a-mediated genome editing.
Conclusion: CRISPR-Cas technology offers precise genome editing capabilities, but controlling its activity is crucial for safe and effective applications. Natural anti-CRISPR proteins and engineered control mechanisms provide valuable tools for modulating CRISPR-Cas activity, enabling researchers to harness its potential while mitigating potential risks.
00:13:28 Mechanisms of Specificity in Anti-CRISPR Proteins
Specificity of Anti-CRISPRs: Anti-CRISPRs exhibit specificity for specific Cas molecules. In the study, anti-CRISPRs 5A1, 5A4, 5A5, BFP, and mCherry were tested against SpiCas9, ASCAS12, and LBCAS9. Only ACR5A1 efficiently inhibited the function of ASCAS12, while all three anti-CRISPRs inhibited LBCAS9.
Mechanism of ACR5A4: ACR5A4 leads to dimerization of Cas12a, blocking double-stranded DNA access and inhibiting its ability to edit.
Mechanism of ACR5A1: ACR5A1 is an enzyme that cleaves the target recognition sequence of the guide RNA. This cleavage is a multi-turnover process, allowing ACR5A1 to continuously inhibit Cas12a.
Mechanism of ACR5A5: The mechanism of ACR5A5 is still unknown.
00:16:00 Bioengineering Strategies for Expanding Cas9 Applications
Permuted Cas9: A study revealed that ACR5A5 functions as an acetyltransferase that modifies Cas12 itself at lysine 635, blocking protospacer adjacent motif recognition. Anti-crispus can be enzymes with distinct mechanisms, affecting either the guide or the protein itself.
Bioengineering Strategies for Cas Enzymes: Challenges of Cas9 include its constant activity and limitations in spatial, temporal, and tissue-specific editing. Circle permutation of Cas9 allows for the development of protease-sensitive Cas9s and the targeted fusion of functional domains.
Structural Considerations for Protein Fusion: The natural N and C termini of Cas9 are far apart from each other and the single-strand DNA, hindering the fusion of protein functional domains. Generating new N and C termini at specific sites on Cas9 can facilitate targeted fusion of functional domains for optimal utility.
00:18:48 Genetic Regulation of Cas9 Activity with Protease Sensors
Building Circle Permutants of Cas9: Circle permutants of Cas9 were constructed by connecting the natural N and C termini with a short linker and then reopening the protein at every other possible option throughout the entire Cas9 molecule. A transposon-mediated library construction method was used to generate a library of these circle permutants. The library was screened functionally using a CRISPR-I assay to identify variants that retained activity.
Identification of Active Circularly Permutated Cas9 Variants: The screen identified a number of circularly permutated Cas9 variants that were still active in both DNA binding and DNA cutting. These variants had rearranged NMC prime termini located in three main regions: the helical two domain, the rough C3 domain, and the CTD.
Circularly Permutated Cas9 Variants Retain Activity in Mammalian Cells: The circularly permutated Cas9 variants retained a significant amount of function in mammalian cells, with some variants showing up to 100% activity compared to wild-type Cas9.
Development of Regulatable Cas9 Enzymes: By shortening the linker between the old N and C prime termini, an inactive or vigilant circularly permutated Cas9 can be generated. A protease target site can be introduced into the linker, allowing the Cas9 molecule to be activated by the presence of a specific protease. This concept of regulatable Cas9 enzymes allows for targeted DNA editing in response to specific pathogens or cellular conditions.
Testing Regulatable Cas9 Enzymes in Bacteria and Mammalian Cells: In bacteria, regulatable Cas9 enzymes were shown to be inducible by specific proteases, including plant proteiviruses and flaviviruses. In mammalian cells, a reporter assay was developed to test the leakiness and activation of regulatable Cas9 enzymes. The regulatable Cas9 enzymes showed minimal leakiness and were activated by flaviviral proteases, leading to DNA double strand breaks.
Single Molecule Sensor Effector for Viral Detection: The regulatable Cas9 enzymes can be used as single molecule sensor effectors to detect the presence of viruses. The system can be genetically encoded within mammalian cells and activated by viral proteases, leading to DNA cleavage and potential defense mechanisms against the virus.
00:26:54 Protein-Level Activation of the CRISPR-Cas9 System
Mechanism of Two-Subunit dCas9 Genome Editing System: The virginal procrease 9 (dCas9) is sterically hindered by a linker, preventing its activity. A protease cleaves the linker, generating a two-subunit cast complex that retains its genome editing capability. Western blotting analysis confirms the formation of the two-subunit complex in the presence of the matching protease.
Altruistic Defense System Using Two-Subunit dCas9: The system was tested in HEK cells with different cell lines expressing various sgRNAs. Targeting a non-essential gene (olfactory receptor) had no significant effect on cell viability. Targeting an essential gene (RPA1) resulted in cell depletion over time, demonstrating the system’s ability to eliminate cells with specific genetic alterations. Targeting a highly amplified locus using sgCyte sgRNA showed the system’s potential for targeting specific genomic regions.
00:29:16 Circular Permutation of Cas Enzymes for Tissue-Specific Editing and Modular Fusion Proteins
Introduction: Cas9 enzymes have been widely used for genome editing, but their natural form has limitations in mammalian systems due to the complexity of DNA repair pathways. Circular permutation of Cas9 can redistribute NNC termini, allowing for the generation of new fusion proteins with different properties.
Pro-Cas9 Enzyme: Pro-Cas9 is an inactive Cas9 enzyme that can be activated by a protease, making it a conditionally active system. Pro-Cas9 can be used for cell autonomous effectors, molecular recording, and tissue-specific activation of Cas9.
Circular Permutant Cas9 as a Scaffold: Circular permutant Cas9 can be used as a scaffold for modular fusion proteins, including DNA modifying enzymes and epigenetic modifiers. This approach can be used to create new base editors with different properties, such as shifted windows of deamination.
Advantages of Circular Permutant Cas9: Circular permutant Cas9 can overcome the limitations of natural Cas molecules in mammalian systems. It allows for the generation of new fusion proteins with specific properties, expanding the applications of Cas9 in genome editing.
Conclusion: Circular permutation of Cas9 enables the generation of new fusion proteins with different properties, expanding the applications of Cas9 in genome editing. This approach has the potential to improve the efficiency and specificity of genome editing in mammalian systems.
Abstract
Revolutionizing Gene Editing: The Transformative Power of CRISPR-Cas and Its Evolving Landscape
In the rapidly advancing field of molecular biology, the CRISPR-Cas system stands as a monumental breakthrough, revolutionizing our approach to gene editing and precision medicine. Initially discovered as a natural defense mechanism in bacteria, CRISPR-Cas has evolved into a versatile platform for drug development, disease modeling, and potential therapies. This article delves into the intricate workings of CRISPR-Cas, including its transformative role in biology, the pivotal contributions of scientists like Jennifer Doudna, and the latest innovations in controlling its activity and enhancing its specificity, such as the use of anti-CRISPR proteins and the bioengineering of Cas9 for improved functionality.
Segment Summaries and Expansion:
CRISPR-Cas: A Revolutionary Tool for Molecular Biology and Precision Medicine
CRISPR-Cas, a transformative tool in biomedicine and biology, enables precise DNA manipulation, rewriting the genetic code to potentially cure diseases at their root. Its diverse applications cover drug discovery, disease modeling, and safety assessment, highlighting its potential for future therapies. CRISPR-Cas enables precise manipulation of genetic information, enabling us to study disease models, investigate single nucleotide polymorphisms, conduct drug screenings, assess safety and toxicology, develop cellular therapies, and explore potential therapeutics for various diseases.
CRISPR-Cas: A Versatile Platform with Broad Applications
This technology significantly impacts drug development, from basic research to clinical trials. It enhances understanding of disease mechanisms and fosters the development of targeted therapies, extending to cellular therapies for a range of diseases. The development of CRISPR-Cas as a genome engineering tool by Jennifer Doudna and numerous collaborators worldwide has revolutionized our understanding and approach to biology, biomedicine, and agricultural technologies. This adaptive immune system from bacteria has been adapted as a powerful tool for biomedicine, recognized by the Dutton Prize awarded to Jennifer Dutton.
CRISPR-Cas System: An Adaptive Immune Defense Mechanism in Bacteria
CRISPR and Cas form a bacterial immune system, protecting against bacteriophages through a process involving adaptation, biogenesis, and interference. This natural mechanism laid the foundation for its applications in gene editing. CRISPR-Cas is an adaptive immune system in bacteria that protects against bacteriophages through a three-step mechanism: adaptation, CRISPR biogenesis, and interference. Cas9, a key Cas protein, is guided by a single guide RNA to target specific DNA sequences and induce double-strand DNA cleavage. Double-strand DNA breaks can be repaired via non-homologous end joining (NHEJ) or homologous recombination (HR), enabling gene knockout or precise genome editing.
CRISPR-Cas9: A Powerful Genome Editing Tool
Cas9, a key protein, and guide RNA work together to enable targeted DNA cleavage. This system utilizes DNA repair mechanisms like non-homologous end joining and homologous recombination, allowing for gene editing and insertion of new genetic information.
Controlling CRISPR-Cas Activity: Natural and Engineered Approaches
Natural control of CRISPR-Cas activity includes anti-CRISPR proteins produced by phages. Engineered approaches involve using these anti-CRISPRs to modulate activity in mammalian cells, with specific examples like ACR5A1, 5A4, and 5A5 inhibiting Cas12a activity. Anti-CRISPR proteins are natural phage-derived molecules that can regulate CRISPR-Cas activity. ACR5A1, 5A4, and 5A5 are anti-CRISPR proteins targeting Cas12a. Identification of anti-CRISPRs was achieved through computational analysis of self-targeting genomes and subsequent in vitro validation. Anti-CRISPRs protect Cas12a proteins from different bacterial origins, providing a versatile tool for controlling CRISPR-Cas activity.
Reporter Assay for Cas Activity:
The HEK-RT1 cell line, a HEK293-based line with a doxycycline-inducible GFP reporter, helps assess Cas12a RNP complex activity through lentiviral transduction of anti-CRISPR molecules and doxycycline induction, visualizing edited cells.
Specificity and Inhibition of Anti-CRISPRs
Anti-CRISPRs exhibit specificity towards certain Cas molecules. ACR5A1 effectively inhibits ASCAS12, while others show varied inhibition efficiencies. These mechanisms involve dimerization of Cas12a and cleavage of guide RNA sequences, showcasing diverse approaches to inhibit Cas activity. Anti-CRISPRs exhibit specificity for specific Cas molecules. In the study, anti-CRISPRs 5A1, 5A4, 5A5, BFP, and mCherry were tested against SpiCas9, ASCAS12, and LBCAS9. Only ACR5A1 efficiently inhibited the function of ASCAS12, while all three anti-CRISPRs inhibited LBCAS9.
New Findings on Anti-CRISPRs and Cas9 Bioengineering
Recent studies reveal diverse mechanisms of anti-CRISPR proteins, targeting either the guide RNA or the Cas protein itself. These findings are crucial for developing new bioengineering approaches to control and enhance Cas9 functionality. ACR5A4 leads to dimerization of Cas12a, blocking double-stranded DNA access and inhibiting its ability to edit. ACR5A1 is an enzyme that cleaves the target recognition sequence of the guide RNA. This cleavage is a multi-turnover process, allowing ACR5A1 to continuously inhibit Cas12a. The mechanism of ACR5A5 is still unknown.
Cas9 Bioengineering for Improved Control and Functionality
Bioengineering approaches aim to overcome limitations of Cas9, such as constant activity and lack of spatial, temporal, and cell-specific control. Circle permutation of Cas9 has led to the development of protease-sensitive Cas9s and targeted fusion of functional domains, enhancing versatility and utility.
Circle Permutants of Cas9 with Protease Activation Sites
Circular permutation of Cas9 enzymes creates pro-Cas9s, which are inactive until activated by a specific protease. This allows for precise control over Cas9 activity in different cellular contexts.
Inducible Systems in Bacteria and Mammalian Cells
Inducible systems have been developed to control Cas9 activity in specific tissues or cell types. These systems utilize conditional promoters or protease activation strategies to achieve targeted DNA cleavage.
Single Molecule Sensor Effector
A single molecule sensor effector has been developed that can detect and record viral presence in mammalian cells. This system uses a pro-Cas9 enzyme that is activated by a viral protease, allowing for targeted and specific detection of viral infections.
Mechanism of Action and Application in Altruistic Defense System
When activated by a matching protease, pro-Cas9 cleaves DNA and forms a two-subunit complex. This complex can then be used to target essential genes in specific cell types, leading to cell depletion or recording mechanisms.
Circular Permutation of Cas9
Circular permutation of Cas9 involves connecting the N and C termini of the protein and reopening it at various points. This creates pro-Cas9 enzymes that are inactive until activated by a specific protease.
Base Editors
Base editors utilizing a nicked Cas9 can enable targeted base editing without double-strand breaks. Circular permutant Cas9s have been used to develop new base editors with varied properties, addressing clinically relevant genetic mutations.
Overcoming Limitations of Natural Cas Molecules
The circular permutation approach allows for the creation of Cas enzymes with new functions, optimized for applications in mammalian systems, overcoming the limitations of naturally occurring Cas molecules.
The advancements in CRISPR-Cas technology, exemplified by the development of circular permutant Cas9s and pro-Cas9 enzymes, herald a new era in gene editing. These innovations offer greater precision, specificity, and versatility, opening up unprecedented possibilities in biomedical research and therapeutic applications. The journey of CRISPR-Cas, from a bacterial defense mechanism to a cornerstone of molecular biology, underscores the immense potential of scientific inquiry and collaboration in transforming our understanding and manipulation of the genetic code.
CRISPR-Cas9 is a revolutionary technology that enables precise genome editing, with applications in research, agriculture, and medicine, but its potential for germline editing raises ethical concerns. CRISPR-Cas9 has transformed research and applications, with over 8,400 PubMed entries by February 2018, and ongoing research and debate focus on its responsible use...
CRISPR technology, exemplified by CRISPR-Cas9, has revolutionized biology and medicine, offering opportunities for gene editing, diagnostics, and treatment of genetic diseases, but challenges remain, including off-target effects, ethical considerations, and accessibility. CRISPR-based diagnostics, particularly for COVID-19, offer rapid, sensitive, and cost-effective pathogen detection, while CRISPR-Cas12a shows promise in gene editing...
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, discovered by Jennifer Doudna, revolutionized gene editing, while Alan S. Michaels' contributions to bioengineering laid the foundation for modern biotechnology....
CRISPR-Cas systems are adaptive immune responses in bacteria that protect against viruses by cleaving foreign DNA. CRISPR-Cas9 technology allows for precise genome editing in eukaryotic cells, raising ethical considerations and the need for responsible progress....
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...
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....