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
00:03:10 Natural Control of CRISPR-Cas Activity
00:13:28 Mechanisms of Specificity in Anti-CRISPR Proteins
00:16:00 Bioengineering Strategies for Expanding Cas9 Applications
00:18:48 Genetic Regulation of Cas9 Activity with Protease Sensors
00:26:54 Protein-Level Activation of the CRISPR-Cas9 System
00:29:16 Circular Permutation of Cas Enzymes for Tissue-Specific Editing and Modular Fusion Proteins

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.


Notes by: Simurgh