Growing Up with Science:
The speaker grew up in a small rural town in Hawaii. The speaker’s father was a professor of American literature. The speaker’s father loved shopping in used bookstores.

A Mind-Blowing Book:
One day, the speaker came home from school and found a copy of “The Double Helix” on their bed. The speaker read the book and was amazed by the story of how scientists discovered the structure of the double helix. The book sparked an interest in science and the desire to solve puzzles in biology and chemistry.

Career Path in Science:
The speaker majored in chemistry with a focus on biochemistry in college. The speaker joined the laboratory of Jack Szostak for graduate school. The speaker worked on genetic recombination and the structure and replication of telomeres.

Moving into New Areas of Research:
The speaker’s mentor, Jack Szostak, was moving into new areas of research, including the origin of life. The speaker was excited about the opportunity to explore new frontiers in biology.

Background and Research Focus:
The scholar’s research focuses on the function of RNA molecules in biology, particularly those with independent functions and not just as protein encoders. Early career centered on understanding the molecular structures and functions of catalytic RNAs called ribozymes.

Linking RNA Research to Modern Biology:
The scholar’s research shifted to exploring the role of RNA in controlling information expression in cells, especially through small RNAs like microRNAs. MicroRNAs regulate the flow of information from DNA to proteins by interacting with other RNA molecules.

Introduction to CRISPR Research:
The scholar’s work with microRNAs led to a collaboration with Jill Banfield, who studied bacteria in extreme environments. Banfield’s lab sequenced DNA from bacteria in acidic, high-temperature, and low-temperature environments to identify the types of bacteria and viruses present.

CRISPR Discovery and Jill Banfield’s Research:
Jill Banfield’s research focused on studying CRISPRs (Clusters of Regularly Interspaced Short Palindromic Repeats) in bacterial genomes. CRISPRs are patterns of DNA sequences that contain repeating elements (black diamonds) and unique sequences between these repeats. Some unique sequences within CRISPRs were found to correspond to sequences found in viruses that infect the bacteria.

CRISPR Evolution and Viral Infection:
CRISPRs evolve over time. When bacteria are isolated and sequenced at different time points, changes are observed in the CRISPR loci. These changes involve the acquisition of new spacer sequences, which are unique sequences derived from viruses that infect the bacteria.

Possible Role of CRISPRs in Bacterial Immunity:
The connection between CRISPRs and viral infection suggests a possible role for CRISPRs in bacterial immunity. CRISPRs may function as a defense mechanism against viral infections by targeting and neutralizing viruses that attempt to infect the bacteria.

CRISPRs as a Novel Gene-Editing Tool:
CRISPRs, particularly the CRISPR-Cas9 system, have been adapted as a gene-editing tool in various organisms. This technology allows for precise targeting and modification of specific DNA sequences, opening up new possibilities for genetic research and therapeutic applications.

Challenges and Ethical Considerations:
Despite the potential benefits of CRISPR-based gene editing, there are challenges and ethical considerations to address. Off-target effects and unintended consequences need to be carefully assessed and minimized. Ethical considerations related to the use of CRISPR technology in human germline editing and its potential long-term implications require careful deliberation and regulation.

Overview:
CRISPR-Cas systems are adaptive immune systems found in bacteria that protect against viruses. CRISPR loci contain integrated sequences from viruses, suggesting an adaptive immune response. CRISPR-associated (Cas) genes are typically found near CRISPR loci and co-vary with their presence.

Mechanism of RNA-Guided Genome Protection:
CRISPR systems transcribe CRISPR loci to produce RNA molecules. Precursor CRISPR RNAs (pre-CRISPR RNAs) are initially formed and contain palindromic repeats that fold into hairpins. These hairpins are recognized by the cell and tag the RNA as CRISPR RNA. Pre-CRISPR RNAs are processed to generate individual molecules containing viral sequences. CRISPR RNAs assemble with Cas proteins to form effector complexes. Effector complexes search for matching sequences in nucleic acids and recruit Cas proteins to cleave the viral DNA.

Anti-CRISPRs:
Phage can fight back against CRISPR systems using anti-CRISPR molecules. Anti-CRISPRs are being studied to understand their mechanisms of action and potential applications.

Responsible Progress:
Ethical considerations are important in the development of CRISPR technology. Scientists should work together to promote responsible progress and minimize potential risks.

Diversity of CRISPR Systems:
CRISPR systems are diverse, with class I systems requiring multiple Cas proteins and class II systems having a single Cas gene for RNA-guided genome protection.

Study of Streptococcus pyogenes’ CRISPR System:
Research on small RNAs in Streptococcus pyogenes led to the discovery of the CRISPR RNA and tracer RNA, which are involved in CRISPR-mediated protection.

Collaboration to Study Cas9:
A collaboration between researchers in Vienna, Sweden, and California aimed to understand the function of Cas9, a protein involved in the CRISPR system of Streptococcus pyogenes.

Discovery of Cas9 as an RNA-Guided DNA Cutter:
Biochemical experiments revealed that Cas9 is an RNA-guided DNA cutter, using a dual RNA interaction to form a functional complex that recognizes DNA molecules. Cas9 unwinds the DNA and triggers a double-stranded DNA break using two separate active sites in the enzyme.

Determination of Active Sites for DNA Cutting:
Mutations in the active sites of Cas9 allowed researchers to determine which site cuts each strand of the DNA.

Mechanism of CRISPR-Cas9 System:
CRISPR-Cas9 utilizes RNA-guided DNA binding to target specific DNA sequences. Active sites of Cas9 are responsible for DNA cutting, and mutations in these sites maintain RNA-guided DNA binding but abolish DNA cutting.

Single Guide RNA (sgRNA):
Simplified system using a single guide RNA (sgRNA) combining CRISPR RNA and tracer RNA. sgRNA enables recognition and cutting of target DNA sequences.

Applications of CRISPR-Cas9 System:
Viral Defense: CRISPR-Cas9 protects bacteria from viral infections by acquiring foreign DNA into the CRISPR sequence and targeting viral DNA for degradation. Genome Editing: In eukaryotic cells, CRISPR-Cas9 can induce double-stranded breaks in genomic DNA, leading to DNA repair with potential changes at the break site. This allows for targeted genome modifications.

The Revolutionary Discovery of Cas9:
Cas9, a programmable protein, revolutionized genome engineering by enabling precise and efficient DNA editing. Unlike previous protein-DNA interactions, Cas9 employs RNA-DNA hybridization for DNA recognition, allowing versatility in genome editing experiments.

Mechanism of DNA Recognition and Unwinding:
Cas9 undergoes significant structural changes upon assembly with guide RNA and DNA. The unwinding of DNA is facilitated by the swinging motion of the HNH domain, enabling the cleavage of the targeted DNA strand. The active structure of Cas9 is achieved only upon engagement with DNA that base pairs with the guide RNA.

Investigating the Dynamics of Cas9 Conformational Rearrangement:
Researchers employed FRET (Fluorescence Resonance Energy Transfer) to study the conformational changes of Cas9 in real-time. By tethering Cas9 to a slide and flowing in different DNA substrates, they monitored changes in FRET interactions. Data revealed that fully matched DNA substrates induced the most significant conformational changes in Cas9, indicating the adoption of the active state.

Implications for Technological Applications:
Understanding the mechanism of Cas9’s DNA recognition and unwinding can guide the development of improved genome editing tools. Insights into the conformational dynamics of Cas9 provide valuable information for designing more efficient and targeted gene editing strategies.

Cas9 Confirmation and States:
Cas9 activity is confirmed through a FRET scale that shows a fully active state when there is no mismatch between the DNA and the guide RNA. Introducing a single base pair mismatch leads to an intermediate state, where the HNH domain (cutting domain) partially swings into place. A fully inactive state is observed when there are multiple mismatches.

Conformational Checkpoint:
The conformational checkpoint allows Cas9 to determine if it is engaged with a fully matched DNA sequence, indicating a potential target for cutting. Mismatched sequences trap Cas9 in the intermediate state, preventing efficient cutting and promoting dissociation from the DNA.

Accuracy of DNA Recognition and Cutting:
The conformational checkpoint ensures accuracy in DNA recognition and cutting, preventing indiscriminate cutting of DNA. This mechanism evolved in response to the constant adaptation of phage to bypass CRISPR systems.

Anti-CRISPR Proteins:
Phage have evolved anti-CRISPR proteins that can interfere with the CRISPR system. These proteins, discovered by Alan Davidson and Joe Bondi, can disable Cas9, providing a defense mechanism for phage.

Mechanisms of Anti-CRISPR Proteins:
In the presence of a prophage containing a sequence recognized by the CRISPR system, bacteria encode anti-CRISPR proteins to prevent CRISPR-mediated cleavage. Anti-CRISPR proteins are small proteins, typically less than 100 amino acids in length, that can shut down the CRISPR system.

Research Approach:
Studying anti-CRISPR proteins involves asking questions and designing experiments to answer those questions. One of the initial questions was to compare different examples of anti-CRISPR proteins and their functions.

Discovery of a Broad-Spectrum Inhibitor of Cas9 Enzymes:
Certain anti-CRISPR proteins, such as C1, C2, and C3, were found to exhibit broad-spectrum inhibition of Cas9 enzymes. This discovery was made through biochemical experiments conducted using purified anti-CRISPR proteins and Cas9 enzymes.

Phylogenetic Diversity of Cas9 Enzymes:
Cas9 enzymes from different bacteria can be highly divergent, despite sharing some conserved features. This diversity is represented in a phylogenetic tree, which illustrates the relationships between different Cas9 proteins.

Experiments with Anti-CRISPR Proteins:
Experiments conducted with purified anti-CRISPR proteins, such as C1, C2, and C3, showed their ability to inhibit biochemical reactions involving Cas9 enzymes. These experiments involved adding anti-CRISPR proteins to reactions containing purified Cas9 enzymes and observing their impact on enzyme activity.

Future Directions:
Ongoing research aims to understand how anti-CRISPR proteins interact with Cas9 enzymes at the molecular level. Further studies are needed to explore the potential applications of anti-CRISPR proteins in biotechnology and genome editing.

Mechanism of Inhibition:
Anti-CRISPR protein C1 inhibits all three Cas9 proteins except strep pyogenes Cas9, which is distantly related. Anti-CRISPR proteins C2 and C3 inhibit only one type of Cas9.

Biochemical Experiments:
C1 blocks DNA cutting by Cas9. C1 traps Cas9 in an inactive but DNA-bound state.

Truncated Proteins:
Truncated forms of Cas9 were used to determine the binding site of the anti-CRISPR protein.

Summary of Binding Mechanisms:
Research revealed a common feature among protein truncations that could bind to C1 anti-CRISPR – the presence of the HNH catalytic domain. The HNH domain binds directly to the C1 protein, as confirmed by separation of the C1 complex bound to the HNH domain. Crystallographic analysis revealed that the anti-CRISPR binds to the active site of the HNH domain, blocking the chemistry necessary for DNA cutting.

Broad-Spectrum Inhibition:
The binding of the anti-CRISPR to the active site explains its broad-spectrum inhibition of Cas9 due to the conservation of this region across Cas9 enzymes. This conserved chemical reaction remains constant over evolutionary time, making the anti-CRISPR a potent inhibitor.

Conformational Inhibition:
The anti-CRISPR blocks the rotation of the HNH domain into position for DNA cutting, taking advantage of the conformational flexibility of Cas9.

Potential Applications:
The inactive DNA-bound complex may be used by bacteria to harness Cas9 for DNA regulation, trapping the protein at sites dictated by the guide RNA.

Ethical Considerations:
The pace of genome editing research is incredibly fast, raising ethical concerns. Examples of recent applications include altering genes in tomatoes to prevent branching and breaking of fruit, which would have been difficult using classical breeding methods.

Applications of Gene Editing:
Precision gene editing enables targeted changes to DNA, leading to potential applications in various fields. A recent study in China used Cas9 to efficiently remove HIV sequences from mouse genomes, showcasing the technology’s potential in treating viral infections.

Germline Editing:
Germline editing involves altering DNA in fertilized eggs or embryos, resulting in heritable changes that can be passed on to offspring. While gene editing in animals has been conducted, the application in humans remains a complex and ethically challenging topic.

Ethical Considerations and Responsible Use:
The National Academies of Sciences and Medicine in the US published a report encouraging research on human genome editing, including in embryos. However, the report also recommended a temporary pause on clinical use in humans until safety and ethical implications are thoroughly evaluated.

Scientist’s Role in Responsible Progress:
Scientists have a significant responsibility to engage in discussions about responsible progress and ensure ethical considerations are at the forefront of gene editing research.

Collaboration and Funding:
Collaboration among researchers and funding support are essential for successful gene editing research and advancements in the field.