Background of CRISPR Technology:
CRISPR technology emerged from the discovery of repetitive DNA sequences in bacteria called CRISPRs (Clusters of Regularly Interspaced Short Palindromic Repeats). These CRISPR arrays contain unique sequences derived from DNA found in viruses, indicating a potential acquired immune system in bacteria. CRISPR-associated (Cas) genes, typically located near CRISPR arrays, encode proteins that are part of this adaptive immune system.

Mechanism of CRISPR-Cas Systems:
CRISPR-Cas systems function as a targeted DNA cutting mechanism in bacteria. They protect cells from viruses by acquiring DNA from invading viruses and integrating it into CRISPR arrays. These viral sequences serve as templates for RNA molecules that combine with Cas proteins to form surveillance complexes. These complexes scan the cell for matching DNA or RNA sequences and induce double-stranded breaks at those sites.

Harnessing CRISPR for Gene Editing:
The targeted DNA cutting activity of CRISPR-Cas systems can be harnessed for gene editing in eukaryotic cells, such as plants and animals. By introducing CRISPR-Cas systems into these cells, precise changes can be made to the genome. This allows for targeted gene editing, insertion, or deletion of genetic material, enabling a wide range of research and therapeutic applications.

New Discoveries and Applications:
Ongoing research continues to uncover new CRISPR pathways and their interactions with other proteins. Anti-CRISPR proteins, found in some cells and phages, can inhibit CRISPR pathways and have potential applications in technology. CRISPR-Cas systems have been adapted for various applications, including gene regulation, disease modeling, and potential therapeutic interventions.

Societal Implications of CRISPR Technology:
CRISPR technology raises important societal and ethical considerations due to its potential to modify the DNA of organisms and even human evolution. It highlights the need for responsible and cautious use of this powerful technology, considering the potential consequences and implications for individuals, society, and the environment.

The Complexity of CRISPR Systems:
CRISPR systems are classified into two major groups: Class I systems involve large protein complexes, while Class II systems utilize simpler single proteins.

Initial Challenges in Harnessing CRISPR Technology:
Early research focused on Class I systems, which required multiple proteins and complex assembly, making them difficult to adapt as a technology.

The Discovery of Single-Protein CRISPR Systems:
Researchers simplified CRISPR technology by identifying Class II systems, which featured a single protein that combined with RNA to perform its function.

Exploring the Function of Cas9:
Jennifer Doudna and colleagues aimed to understand the role of Cas9, a key protein in Class II CRISPR systems, in protecting cells from viral infection.

Cas9’s Remarkable DNA-Cutting Ability:
Cas9 was found to be an enzyme that can recognize double-stranded DNA at specific positions matching a 20-nucleotide sequence in the guiding RNA.

Single Guide RNA:
A single guide RNA was created by combining the natural CRISPR RNA and tracer RNA, simplifying the system and making it more accessible for researchers.

Precise Genome Editing with Cas9:
The single guide RNA guides Cas9 to a specific site in the genome, enabling precise double-stranded breaks in DNA, a crucial step for genome editing.

Cas9 Protein Function in Eukaryotic Cells:
Cas9 protein is unique in its ability to cleave DNA without hydrolyzing ATP or GTP. Understanding its mechanism of action is crucial for harnessing its DNA recognition capabilities in various applications.

Cas9 Conformation Changes upon Nucleic Acid Binding:
Cas9 undergoes conformational changes upon binding to nucleic acids. These changes reorient the protein to open up a channel for the guide RNA and accommodate the RNA-DNA hybrid formation.

Cas9-DNA Interactions and Substrate Specificity:
Cas9 protein needs to unwind DNA without external energy sources. The HNH domain in Cas9 acts as a chemical cleaver, cutting the DNA strand complementary to the guide RNA.

Importance of the Non-Target Strand in Cas9 Activity:
The presence of the non-target strand influences the conformation of Cas9. It triggers the HNH domain to move into the correct position for DNA cleavage.

Investigating Cas9 Conformational Changes:
Biochemical assays are employed to study conformational changes in Cas9. Changes in distances between amino acids can indicate structural rearrangements.

Cas9 Sensitivity to Target DNA Sequence:
Cas9 activity is highly sensitive to the level of base pairing between the guide RNA and target DNA. Close but imperfect matches often do not result in chemical alterations of the DNA.

Monitoring Cas9 Structural Changes:
Researchers introduced chemical dye pairs on Cas9 to monitor conformational changes. In the inactive state, the dyes are far apart and show low fluorescence resonance energy transfer (FRET) signal. Upon DNA binding, the dyes move closer, resulting in a strong FRET signal. This setup allows for real-time monitoring of Cas9 interactions with DNA.

Intermediate Conformation:
Cas9 can adopt an intermediate conformation between inactive and active states. The population of this intermediate state depends on DNA-guide RNA complementarity. This intermediate state suggests that Cas9 acts as a sensor, detecting the degree of match between target DNA and guide RNA.

Designing Improved Cas9 Variants:
Ongoing efforts aim to design Cas9 variants with enhanced accuracy and DNA-sensing capabilities. By introducing specific mutations, researchers seek to improve the protein’s performance in gene editing applications.

Anti-CRISPR Mechanisms:
Microorganisms have evolved anti-CRISPR systems to counter CRISPR defenses. Anti-CRISPR proteins, often small proteins under 100 amino acids, can inhibit CRISPR components. Understanding anti-CRISPR mechanisms provides insights into the function of CRISPR proteins, particularly Cas9.

Unpublished Data on Anti-CRISPR Protein C1:
Anti-CRISPR protein C1 can block Cas9 activity. Structural and biochemical studies are underway to elucidate the mechanism of C1 inhibition. Investigating anti-CRISPR mechanisms can inform the design of more effective CRISPR-based technologies.

Biochemical testing of Cas9 cleavage:
Using a radioactive label on one strand of a double-stranded DNA target sequence, Cas9 enzyme cleaves the DNA efficiently. Adding an inhibitor blocks DNA cleavage, indicating the inhibitor’s involvement in the process.

Distinguishing between binding and cleavage inhibition:
A non-denaturing gel system is used to visualize protein trapping during DNA association. Without an inhibitor, increasing Cas9 and guide RNA leads to DNA cutting and faster migration on the gel. Adding the inhibitor prevents DNA cutting and traps radiolabeled DNA with Cas9 and the guide RNA, indicating binding but no cleavage.

Investigating the mechanism of inhibition:
Variants of the Cas9 protein are created to understand the role of the HNH domain. HNH domain inclusion in the construct results in DNA cleavage inhibition, while its removal allows cleavage.

Conclusion:
The experiments reveal that the inhibitor doesn’t prevent Cas9 binding to DNA but specifically blocks DNA cleavage. The HNH domain of Cas9 plays a crucial role in this inhibition, suggesting a potential mechanism for controlling Cas9 activity.

Protein-Protein Interaction and Inhibition:
Experiments revealed binding between the HNH domain of Cas9 and an anti-CRISPR protein. Chimeric constructs confirmed that the HNH domain is responsible for this interaction. Crystallographic analysis showed that the inhibitor physically blocks the chemically important residues in the HNH domain, preventing DNA interaction.

Potential Technological Application:
Scientists can create inactivated Cas9 variants by modifying critical residues, including those in the HNH domain. By linking effector proteins to these inactivated Cas9 variants, researchers can regulate gene expression without DNA cleavage.

Anti-CRISPR Proteins in Phage and Bacteria:
Phage or bacteria may use anti-CRISPR proteins to regulate Cas9 activity. By preventing DNA cutting while maintaining RNA-guided binding, anti-CRISPR proteins could expand the natural functions of Cas9 in cells.

Responsible Progress and Applications:
The rapid adoption of CRISPR technology has led to its use in over 100 organisms and cell types. Recent examples include targeted genetic modifications in tomatoes for stronger branches and the removal of HIV proviruses from mouse genomes. Germline editing raises ethical concerns and has prompted discussions on appropriate regulations and guidelines.

Importance of Fundamental Research Funding:
Fundamental research, including the work on CRISPR, is crucial for technological advancements and economic growth. Reduced funding for research could hinder the U.S.’s ability to compete in these areas. Communicating the importance of science funding to the public and policymakers is essential.

Modulating Cas9 Activity:
Researchers are actively developing techniques to regulate Cas9 enzymes in cells. Engineered proteins and natural components are being utilized to enhance safety and accuracy. Anti-CRISPRs are employed to limit Cas9 activity at off-target sites, improving specificity.

CRISPR Adaptation:
Adaptation involves acquiring new viral sequences into CRISPR arrays. A CRISPR integrase complex, consisting of six protein copies, precisely integrates DNA fragments into the CRISPR array. In E. coli K12, a DNA binding protein contributes to the accuracy and efficiency of the integration process.

Potential Applications:
George Church’s lab has harnessed the integrase as a technology for cell recording, integrating DNA bits into cell populations, similar to natural mechanisms. Unexplored aspects of CRISPR systems hold promise for future applications, leveraging natural mechanisms, including adaptation.

Cas9 Inhibition:
Inhibiting one domain of Cas9 affects cutting of the other DNA strand, indicating a strong coupling between the two domains.