Overview of Jennifer Doudna’s Research Career:
Jennifer Doudna’s research journey has focused on the biology of RNA molecules and their role in controlling genetic information expression. She began her graduate studies at Harvard, where she demonstrated the catalytic potential of RNA in synthesizing a complementary RNA strand, providing biochemical evidence for the RNA world. She then moved to Colorado to work with Tom Cech, where she studied RNA structure, particularly group one self-splicing ribozymes and introns, and helped identify the core of the intron and its structure.

Pioneering Work on RNA Structure and Function:
Doudna’s desire to obtain high-resolution structures of RNA molecules led her to establish her own lab, where she successfully crystallized the group one P4, P6 intron, paving the way for further exploration of complex RNA structures. This breakthrough earned her recognition and honors, including election into the National Academy of Sciences before the age of 40.

Transition to UC Berkeley and Focus on MicroRNAs and RNAi:
Doudna moved her lab to UC Berkeley, where she continued her research on RNA structure and function, investigating viral RNAs and developing an interest in microRNAs and the RNAi pathway. Her work in this area established her as a leading expert in the field of RNA structure and function.

CRISPR Biology and Genome Editing:
Doudna’s groundbreaking work on CRISPR biology and genome editing has revolutionized the field of genetic research. Her pioneering experiments in the world of CRISPR RNA have provided insights into genome editing, recombination, and genome engineering, enabling precise modifications to DNA. This discovery has broad implications for genetic research, disease treatment, and agricultural applications.

Historical Discovery of CRISPRs:
In 1987, a highly repetitive sequence was noticed in bacterial DNA, but its significance was unknown. Around 2005, three papers revealed the presence of CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats) in bacterial chromosomes, with unique sequences between repeat sequences. The unique sequences were found to correspond to sequences found in bacteriophage that could infect those bacteria.

CRISPR as a Bacterial Immune System:
CRISPR loci are involved in a bacterial immune system that defends against phage infections and plasmid transformation. Foreign DNA is detected and small bits are excised and inserted into one end of the CRISPR locus. The CRISPR locus is transcribed into RNA, processed into smaller RNA units, and assembled into complexes capable of recognizing and destroying foreign DNA.

Molecular Mechanism of CRISPR Activation:
CRISPR loci are transcribed as long precursor molecules that can fold back on themselves to form short hairpin-like structures. CRISPR-associated enzymes cleave these sequences to generate small RNAs that are functional in the system. Mature CRISPR RNAs have identical ends derived from the repeats and unique central sequences that can base pair with complementary sequences in double-stranded DNA. These RNAs function in the context of proteins to form complexes that recognize complementary sequences in double-stranded DNA targets.

Structural Insights into Type 1 CRISPR Systems:
CRISPR systems are classified into three major subtypes: type 1, type 2, and type 3. Type 1 systems involve large multi-protein complexes called Cascade that assemble with a CRISPR RNA and recruit Cas3 nuclease to cleave double-stranded DNA. Cascade complex comprises five different proteins (Cas A-E) and a single copy of CRISPR RNA. CasE protein cuts the CRISPR RNA into unit-length molecules and remains bound to the RNA hairpin. Cas-C proteins cradle the RNA and present it for recognition of double-stranded DNA. Recent cryo-EM studies revealed the structure of Cascade bound to double-stranded DNA and the Cas3 endonuclease. The DNA corkscrews into the base of the Cascade complex and opens up for base pairing with the guide RNA sequence. Cas3 protein sits adjacent to the DNA-RNA pairing region and cleaves the DNA.

Type 2 CRISPR Systems:
Type 2 CRISPR systems employ a single protein, Cas9, as a programmable nuclease. Cas9 is guided to a sequence by CRISPR RNA and cleaves DNA in a sequence-specific manner. The discovery of type 2 CRISPR systems opened up new avenues for genome editing and gene therapy. Emmanuelle Charpentier’s research on CRISPR transcripts in Streptococcus pyogenes led to the identification of transactivating RNAs that generate short segments of double-stranded RNA. These double-stranded RNAs are substrates for the host ribonuclease 3 enzyme, which is related to the dicer enzyme involved in RNA interference. The discovery of type 2 CRISPR systems and their potential for genome editing has revolutionized the field of molecular biology.

Cas9’s Role in CRISPR Function:
Cas9 is a dual RNA-guided, double-stranded DNA cleaving enzyme. It requires both CRISPR RNA and tracer RNA to function. Cas9 binds to the CRISPR RNA, which contains the DNA-targeting sequence. The tracer RNA forms a structure with the CRISPR RNA, recruiting Cas9 to the target DNA sequence.

Cas9’s DNA Cleavage Mechanism:
Cas9 has two molecular blades that cut one strand of DNA each, complementary to the CRISPR RNA. This results in a double-stranded break in the DNA.

Collaborative Research:
Jennifer Doudna and Emanuel Charpentier collaborated to study Cas9’s function. Martin Jinek and Christoph Chylinski purified Cas9 and demonstrated its DNA-cleaving activity.

Discovery of the Single Guide RNA Concept:
Martin Jinek identified a minimal functional region within the CRISPR RNA complex. The idea of a single guide RNA was conceived, combining the essential parts of the two natural RNAs. This single guide RNA concept enabled DNA cutting by simply changing the guide RNA sequence.

Programmable DNA Cutting with Cas9:
Martin Jinek designed five guide RNAs targeting different sequences in the GFP gene. Cas9 programmed with these guide RNAs efficiently cut plasmid DNA. Double cutting of the plasmid confirmed the precise targeting of Cas9.

Publication and Initial Experiments:
The research team published their findings in the summer of 2012. Experiments began to adapt the system for use in eukaryotic cells. Nuclear localization signals were added to Cas9 for entry into the eukaryotic nucleus. Artificial genes encoding Cas9 were generated for expression in human cells.

Rapid Expansion of CRISPR Applications:
In early 2013, multiple publications demonstrated Cas9’s ability to make targeted changes in various cells and organisms. Successful applications included human cells, mouse cells, zebrafish, yeast, bacteria, and plants. CRISPR-Cas9 emerged as a powerful genome engineering tool.

Understanding the Mechanism:
Researchers aimed to understand how CRISPR-Cas9 finds and cleaves target sites within vast genomic DNA. Biochemical experiments were conducted using plasmid DNA and DNA oligos as substrates.

DNA Curtains for Visualizing CRISPR-Cas9 Behavior:
Eric Green of Columbia University introduced the concept of DNA curtains. DNA curtains are single molecules of phage lambda DNA used to visualize polymerases and repair enzymes. CRISPR-Cas9 complexes were tested on DNA curtains to observe their behavior.

Collaboration with Eric Green:
A collaboration was initiated between Jennifer Doudna’s lab and Eric Green’s lab. Sam Sternberg, a graduate student, visited New York City to study CRISPR complexes on DNA curtains. Initial experiments investigated the ability of CRISPR-Cas9 to locate and bind to target sites.

Single-Molecule Imaging of Cas9-Lambda DNA Interactions:
Researchers conducted experiments to observe the localization of Cas9 complexes along lambda DNA using quantum dot labeling and single-molecule imaging.

Tethered Lambda DNA Assay:
DNA was tethered on one end and subjected to a flow of buffer to extend or retract the DNA molecules. Quantum dot-labeled Cas9 complexes were visualized and analyzed as they interacted with the DNA.

Localization of Cas9 Complexes:
Cas9 complexes were observed to specifically localize at programmed sites along the DNA. Binding events were observed to be long-lived, indicating stable interactions between Cas9 and the target DNA.

Control Experiments:
Experiments were performed with multiple programmed complexes to validate the assay’s specificity and accuracy.

Collaboration for Functional Studies:
Sam and Cy Redding collaborated to investigate the functional aspects of Cas9 targeting.

Requirement for Cleavage Activity:
Researchers initially used inactive and wild-type Cas9 to study its binding behavior. Unexpectedly, both inactive and wild-type Cas9 showed similar tight binding to target sites.

Testing Cas9’s Activity:
Experiments were conducted to investigate whether Cas9 was actually cleaving DNA or remaining bound to uncleaved DNA. Single-molecule experiments were performed using DNA curtains and Cas9 complexes.

High Salt Buffer:
A buffer with a high salt concentration was used to dissociate any proteins bound to DNA. When the high salt buffer was introduced, many DNA molecules were observed to be cut at the predicted target site.

Doubly Tethered DNA:
A doubly tethered DNA setup was used, where the DNA was attached at both ends. Cas9 bound to the target sequence, and when the high salt buffer was introduced, the DNA swung down, indicating cleavage. Cas9 remained associated with the broken end of the double-stranded DNA.

Stoichiometric Cleavage:
Bulk assays varying the ratio of Cas9 RNA complexes to DNA substrate revealed stoichiometric cleavage of DNA. This observation indicated that Cas9 binds to DNA, cuts it, and remains bound to the product, without turnover.

Denaturing and Non-Denaturing Gel Electrophoresis:
Denaturing polyacrylamide gel electrophoresis showed cleavage of DNA, while non-denaturing gel electrophoresis showed that Cas9 complexes remained assembled. This confirmed that Cas9 forms a stable complex with cleaved DNA ends under these biochemical conditions.

Nonspecific Binding Events:
Cas9 displayed nonspecific binding to DNA at non-target sites, particularly in GC-rich regions of the lambda genome.

Potential Specificity for PAM Sequences:
These nonspecific binding events prompted the hypothesis that Cas9 might specifically recognize PAM sequences (GG dinucleotides).

Competition Experiments:
Sam conducted competition assays to test whether Cas9 interacts with PAM sequences in double-stranded DNA. The higher the density of PAM sequences in the competitor DNA, the better it competed in DNA cleavage reactions.

Target Recognition Mechanism:
Cas9 appears to first recognize a PAM motif and then interrogates adjacent DNA for complementarity to the guide RNA. This mechanism allows Cas9 to efficiently sift through genomic DNA to find bona fide target sites.

Importance of PAM Motif:
A single point mutation in the PAM motif of a DNA substrate resulted in the DNA functioning as a competitor, further emphasizing the importance of PAM recognition. Cas9 cleaves double-stranded DNA with a PAM motif, but not single-stranded DNA complementary to the guide RNA, highlighting the significance of PAM-mediated target recognition.

Mechanism of Cas9 activation:
Cas9 requires a guide RNA and PAM sequence for DNA cleavage. Single-stranded DNA is not a substrate for Cas9, but it becomes one when PAM nucleotides are added. This suggests that PAM recognition allosterically activates Cas9.

Crystal structure of Cas9:
Martin Jinek determined the crystal structure of Cas9 in its apo form (without bound nucleic acids). The structure revealed three motifs: a RecA-like domain, a nuclease domain, and a C-terminal domain. The RecA-like domain binds the guide RNA, while the nuclease domain cleaves the DNA. The C-terminal domain is involved in protein-protein interactions.

Mechanism of DNA cleavage:
Cas9 uses a two-step mechanism to cleave DNA. In the first step, Cas9 binds to the guide RNA and the target DNA. In the second step, Cas9 cleaves the DNA at a site adjacent to the PAM sequence. The two-step mechanism ensures that Cas9 only cleaves DNA at specific target sites.

Structure of Cas9:
Cas9 has two active sites: RuvC and HNH nuclease domains. A large alpha-helical lobe is likely the binding site for the guide RNA handle. A hinge sequence with point mutations affecting DNA targeting is identified.

Cross-linking Experiments:
Cross-linking experiments identified two disordered loops near the potential hinge sequence. Mutations in these loops disrupt DNA binding and cleavage by Cas9. The loops may start in a dynamic state, becoming ordered upon binding to a bona fide PAM sequence.

EM Data:
Negative stain EM reveals that Cas9 rearranges upon binding to nucleic acid ligands, forming a central channel for double-stranded DNA. The ends of the RNA and DNA molecules are visualized in the fully loaded complex.

Conclusion:
Cas9 undergoes conformational changes upon binding to nucleic acid ligands, forming a central channel for double-stranded DNA. The guide RNA and DNA ends are positioned in the complex.

Structural Features of Cas9:
Cas9 has two distinct regions or domains flanking the central channel where the DNA substrate binds. In the apo state, the central channel is not present, but upon association with the guide RNA, a conformational rearrangement occurs, creating a central binding pocket for DNA.

Cas9 as a Tool in Cells:
Cas9 is not only a fascinating research subject but also a promising tool for cellular applications. Understanding Cas9’s mechanism of action will aid in improving its efficiency as a tool and provide insights into how enzymes access DNA, handle chromatin structure, and trigger recombination pathways.

Collaborators and Contributors:
Jennifer Doudna acknowledges the contributions of several individuals to the Cas9 project: Sam Sternberg and Cy Redding for their work on single-molecule and bulk assays. Martin Jinek for initiating the Cas9 project and his ongoing investigations into its mechanism. Emanuel, Christoph, Evan Ogalis, and members of Doudna’s lab for their contributions to the structural biology of Cas9.

Questions from the Audience:
A question is raised regarding the length and sensitivity of the DNA sequence recognized by Cas9.

Target DNA Sequence Length and Tolerance to Mutations:
Cas9 targets a 20 base pair DNA sequence plus a Protospacer Adjacent Motif (PAM), totaling about 22 base pairs. The first 12 base pairs closest to the PAM are most crucial for targeting and poorly tolerate mutations. Mutations farther away from the PAM are more tolerable, allowing for some flexibility in the target sequence.

Engineering Cas9 for Specificity and Applications:
Researchers are exploring other naturally occurring Cas9 variants with different target sequence lengths. Engineering Cas9 variants with increased specificity is a topic of interest. Approaches like using Cas9 as a nickase, requiring two independent recognition events for DNA cleavage, are being investigated to enhance control and specificity.

Cas9 in Archaea:
No known examples of type 2 systems with Cas9 in archaea. Many archaea have CRISPR systems, but they are all of other types, not type 2.

Engineering Cas9 to Target Different PAMs:
Possible to engineer Cas9 or find other Cas9s that recognize different PAM sequences. Nature has already done this, resulting in Cas9 variants that recognize different PAMs. Aim is to understand PAM recognition to engineer Cas9s with different specificity.

Watson-Crick Base Pairing and Target Site Selection:
Importance of Watson-Crick base pairing between RNA and DNA target highlighted. Evidence from cascade complex reconstructions suggests short segments of base pairing between guide RNA and DNA, separated by kinks. Similar binding mode supported by chemical probing experiments.

Double-Stranded DNA Opening:
Opening of double-stranded DNA during strand invasion is ATP-independent. Conformational changes in Cas9 and cascade complexes upon substrate association are observed. Hypothesis that conformational change is coupled to duplex opening. Ongoing research to elucidate the mechanism of double-stranded DNA opening.