Welcome and Housekeeping: Greg Hannon welcomed attendees to the Cold Spring Harbor meeting on regulatory and non-coding RNAs. He emphasized the goal of making Cold Spring Harbor a home for the non-coding RNA community and coordinating meetings with EMBL in Europe. Housekeeping details included talk durations, available microphones for questions, and the availability of talks on the LeadingStrand website.
Meeting Structure: The meeting consisted of keynotes, talks, and posters, with a 50-50 split between presentation formats. A wine and cheese reception was scheduled for the afternoon, and participants were encouraged to visit the bar after 5 pm. Interactive participation was encouraged, with attendees advised to raise their hands for questions and wait for the microphone.
First Keynote Speaker: Michael Turns introduced Jennifer Doudna, a renowned RNA expert from UC Berkeley, as the first keynote speaker. Doudna’s research focuses on combining structural biology and biochemistry to tackle diverse RNA problems.
CRISPR: An RNA-Guided Bacterial Immune System: Doudna discussed her research on CRISPR, a bacterial immune system that uses RNA molecules to guide DNA cleavage. CRISPR loci contain short palindromic repeats and unique spacer sequences that match sequences found in phage and plasmids. CRISPR-associated (Cas) genes encode proteins involved in the CRISPR immune system. When infected by phage or transformed by plasmids, bacteria can acquire short sequences from the foreign genetic material and incorporate them into the CRISPR locus. The CRISPR locus is transcribed and processed to produce shorter molecules that assemble with Cas proteins to form a silencing complex.
CRISPR-Cas as a Defense Mechanism: CRISPR loci provide an effective defense mechanism against foreign nucleic acids, particularly phages. The CRISPR RNA (crRNA) guides the CRISPR-Cas complex to target and degrade foreign DNA molecules that match the crRNA sequence.
CRISPR RNA Processing and Structure: The initial long transcript containing repeated sequences and spacer elements undergoes processing to generate shorter crRNAs. Each crRNA molecule consists of a unique sequence in the middle, flanked by identical sequences derived from the repeat element. The crRNA forms a hairpin-like structure, allowing for base pairing with foreign genetic material.
Effector Complex Assembly and Function: CrRNAs assemble with one or more proteins to form an effector complex. This complex utilizes the crRNA sequence to base pair with foreign genetic material, leading to its degradation. The precise mechanism of action varies among different CRISPR systems.
Diverse CRISPR Systems: There is not a single, universal CRISPR system. A large family of related CRISPR systems exists, exhibiting variations in enzyme composition and mechanisms.
Ongoing Research: Researchers continue to investigate the intricate mechanisms and components involved in CRISPR-mediated immunity. Understanding these systems provides insights into microbial defense strategies and potential applications in biotechnology and medicine.
Diversity of CRISPR-Associated Genes: CRISPR-associated genes (cast genes) show considerable diversity in terms of their color and number of arrows. Sequence-level homology is challenging to establish due to rapid evolution.
Horizontal Transfer of CRISPR Loci: Organisms often possess multiple CRISPR loci, each with its unique type. The rapid exchange of these loci through horizontal transfer drives the evolution of CRISPR systems.
The Role of Small RNAs in CRISPR: Jennifer Doudna’s research focuses on how small RNAs (guide RNAs) are generated in CRISPR systems. These guide RNAs assemble with proteins to destroy specific nucleic acids complementary to the CRISPR RNA.
Collaboration with Emanuel Charpentier: Doudna collaborated with Emanuel Charpentier, a microbiologist from Umea University in Sweden. Charpentier was studying the CRISPR locus in S. pyogenes, a human pathogen.
CRISPR Locus in S. pyogenes: The CRISPR locus in S. pyogenes contains a characteristic set of CRISPR repeats and four cast genes. This system has a smaller number of genes compared to other CRISPR pathways.
00:17:39 CRISPR-Cas9: A Dual RNA Guided DNA Endonuclease
Discovery of Cas9’s Function: Jennifer Doudna and Emanuel Charpentier began collaborating to investigate the function of Cas9, a protein involved in the CRISPR system. Cas9 is a large protein that had been challenging to study due to difficulties in producing a purified version.
CRISPR RNA Production in the Studied System: In the CRISPR locus studied, CRISPR RNAs are generated through a unique process involving: Initial transcription of the CRISPR locus to form a long precursor molecule. Base pairing of the precursor molecule with tracer RNAs to form short duplexes. Cleavage of the duplexes by the host ribonuclease 3 enzyme and Cas9, resulting in mature CRISPR RNAs.
Cas9’s Role in Plasmid Transformation: Cas9 was found to be required for blocking plasmid transformation in cells when the plasmids contained sequences matching CRISPR RNAs. This observation suggested that Cas9 might possess endonuclease activity capable of cutting DNA in an RNA-guided manner.
Dual RNA Guidance of Cas9: Experiments revealed that Cas9 is a dual RNA-guided DNA endonuclease, requiring both a CRISPR RNA and a tracer RNA to function. Cleavage of plasmid DNA by Cas9 was only observed in the presence of both RNAs. Cas9 could cleave both circular and pre-linearized plasmid DNA, indicating that circularization or supercoiling was not necessary for cleavage.
Significance of the Discovery: This discovery provided strong evidence supporting the hypothesis that Cas9 is an endonuclease capable of RNA-guided DNA cleavage. It paved the way for further research and development of Cas9 as a versatile gene-editing tool, revolutionizing the field of genome engineering.
Cas9 Cleavage Sites: Cas9 creates double-strand breaks (DSBs) at specific target sites in DNA. These cleavage sites occur upstream of the end of the CRISPR RNA-target DNA base pairing region.
PAM Sequence: The Proto Spacer Adjacent Motif (PAM) is a short DNA sequence essential for target recognition by Cas9. It is located immediately downstream of the CRISPR RNA-target DNA base pairing region.
Tracer RNA Interactions: A tracer RNA can base pair with a sequence in the CRISPR RNA adjacent to the target-binding region. This interaction forms a complex between the two RNAs, the two DNA strands, and the Cas9 protein.
Cas9’s Endonuclease and Exonuclease Activities: Cas9 exhibits both endonuclease and 3′ to 5′ exonuclease activities. It rapidly cleaves both DNA strands at the target site, followed by further trimming of the non-complementary strand from the 3′ end.
Multiple Turnover Reaction: Cas9 can cleave multiple DNA molecules per complex with CRISPR RNA. This indicates that the Cas9-RNA complex undergoes multiple rounds of target recognition and cleavage.
Simplified Cartoon of Cas9 Protein: A simplified representation of the Cas9 protein is provided for further understanding of its structure and function.
00:25:28 Molecular Mechanisms of Cas9-Mediated DNA Cleavage and Target Binding
Cas9 Catalytic Domains: Cas9 has two known nuclease domains: the RUBC and HNH nuclease domains. Mutations in either of these domains result in nicking of supercoiled DNA instead of full cleavage. The HNH nuclease domain is essential for cleaving the complementary strand, while the Rov-C domain is essential for cleaving the non-complementary strand.
Tracer RNA: Tracer RNA is essential for target binding and possibly for binding and orienting the CRISPR RNA. Without the tracer RNA, there is very little formation of any complex between Cas9 and DNA. The tracer RNA does not have any complementarity to the targeted DNA.
Cas9 Cleavage Mechanism: Cas9 binds to a complex of CRISPR RNA and tracer RNA, forming a larger RNP. The CRISPR RNA sequence base pairs with one strand of the DNA sequence, leading to its cleavage by the HNH and Rov-C active sites in Cas9. Cleavage occurs three base pairs upstream of the PAM motif. Each nuclease domain cleaves one strand of the DNA.
00:30:42 CRISPR-Cas9 Target DNA Requirements for Cleavage
PAM Motif: CRISPR systems require a short sequence motif, known as the PAM (protospacer adjacent motif), adjacent to the sequence recognized by the CRISPR RNA. In the system being studied, the PAM motif consists of just two base pairs on the wild type strata GG. Mutations in either or both of the Gs of the PAM motif abolish cleavage and binding of the CRISPR RNA to the target DNA.
Plasmid Transformation: Plasmids with a wild-type PAM sequence and targeted by the CRISPR system yield very few transformants. Mutations in the PAM motif restore transformation efficiency, indicating the importance of the PAM motif for CRISPR-mediated interference.
DNA Oligonucleotides and Binding Assay: DNA oligonucleotides with mutated PAM nucleotides show no cleavage or binding by the CRISPR RNA. This demonstrates the critical role of the PAM motif in CRISPR-mediated DNA targeting and binding.
Minimal Requirements for CRISPR and Tracer RNAs: The researchers are investigating the minimal requirements for CRISPR and tracer RNAs in the system.
00:33:30 CRISPR-Cas9: A Programmable DNA Endonuclease Guided by RNA
Chimeric RNA Design: Researcher Martin Jinek created a single transcript, or chimeric RNA, that encompassed all the necessary functionalities for targeting and cleavage. This chimeric RNA included the part of the CRISPR RNA that binds DNA and the part of the tracer RNA that interacts with the CRISPR RNA and associates with Cas9 protein. Capping the ends of the RNAs with a GA3 tetraloop formed a chimeric transcript about 60 nucleotides in length.
Cleavage Efficiency: Chimera A, with a slightly longer extension on the three prime end, showed efficient DNA cleavage comparable to the natural sequence. Chimera B, truncated by about 10 nucleotides on the three prime end, exhibited no observable cleavage.
Programmable DNA Cleavage: Martin designed five chimeric RNAs to target specific sites on a plasmid encoding GFP. All five chimeric RNAs successfully cleaved the plasmid DNA, demonstrating the programmability of the system.
Verification of Cleavage Sites: To confirm the accuracy of cleavage, a restriction site near the targeted regions was used to verify that cleavage occurred at the predicted sites.
Potential for Genome Editing: The programmable DNA endonuclease guided by RNA offers advantages over existing genome editing methods, such as TALON proteins and zinc finger nucleases. Changing the sequence of a small RNA can retarget the system to different sites, eliminating the need for a new protein for each target.
Collaborative Effort: The successful development of the system was a result of collaboration between Jennifer Doudna’s lab and Emanuel Charpentier’s lab. Researchers from both labs contributed to the project, including Martin Jinek, Mickey Hauer, Rachel, Kai Hong, and Sam.
Unwinding of Double-Stranded DNA: The mechanism by which double-stranded DNA is unwound during cleavage is not yet fully understood. It is hypothesized that recognition of the PAM sequence triggers a conformational change in the effector complex, leading to local unwinding of the duplex.
00:44:02 CRISPR-Cas9: Mechanisms, Variants, and Applications
Mechanism of PAM Recognition: The CRISPR-Cas9 system only cuts DNA sequences flanked by a protospacer adjacent motif (PAM), a specific DNA sequence. This PAM recognition is crucial for the system’s ability to target specific DNA sequences. The molecular details of PAM recognition are not yet fully understood, but understanding this mechanism could open up more target sites in the system.
Targeting Stability and Turnover: The stability of the CRISPR-Cas9 complex on the DNA target raises questions about how turnover works and how new substrates are accessed. The thermodynamics of the complex may play a role in targeting efficiency and turnover.
Cross-Reactivity of Cas9 Variants: Different Cas9 variants from various organisms can cross-react with each other’s dual RNAs, enabling the system to target diverse DNA sequences. The ability of Cas9 variants to bind to different RNA structures provides opportunities for expanding the target range of the system.
Effect of Single Point Mutations on Binding and Cleavage: Single point mutations in the CRISPR-Cas9 system may affect cleavage activity but not necessarily binding. These mutations could result in a stable complex formation without cleavage, suggesting that all necessary components for binding are still present.
Phage Avoidance of PAM Sites: Bacteriophages, viruses that infect bacteria, try to avoid having PAM sites in their genomes to escape the CRISPR-Cas9 system. Phages mutate their PAM sites to evade the CRISPR system, highlighting the effectiveness of this defense mechanism.
Abstract
Revolutionizing Gene Editing: Insights from the Cold Spring Harbor Meeting on Regulatory and Non-Coding RNAs
Greg Hannon warmly welcomed attendees to the Cold Spring Harbor meeting on regulatory and non-coding RNAs, expressing his aspiration to establish Cold Spring Harbor as a hub for the non-coding RNA community and to foster collaboration with EMBL in Europe. Housekeeping details, such as talk durations, microphone availability, and access to presentations on the LeadingStrand website, were also communicated.
The meeting structure consisted of keynotes, talks, and poster presentations, with an even balance between these formats. A wine and cheese reception was scheduled for the afternoon, and participants were encouraged to socialize at the bar after 5 pm. Interactive participation was highly encouraged, with attendees advised to raise their hands for questions and await the microphone.
The first keynote speaker, Jennifer Doudna, an esteemed RNA expert from UC Berkeley, was introduced by Michael Turns. Doudna’s research delves into the combination of structural biology and biochemistry to tackle diverse RNA problems.
Doudna presented her research on CRISPR, a bacterial immune system that employs RNA molecules to guide DNA cleavage. CRISPR loci contain short palindromic repeats and unique spacer sequences that match sequences found in phage and plasmids. CRISPR-associated (cast) genes encode proteins involved in the CRISPR immune system. Upon infection by phage or transformation by plasmids, bacteria can acquire short sequences from the foreign genetic material and integrate them into the CRISPR locus. The CRISPR locus is then transcribed and processed to produce shorter molecules that assemble with Cas proteins to form a silencing complex.
CRISPR loci serve as an effective defense mechanism against foreign nucleic acids, particularly phages. The CRISPR RNA (crRNA) guides the CRISPR-Cas complex to target and degrade foreign DNA molecules that match the crRNA sequence. The initial long transcript containing repeated sequences and spacer elements undergoes processing to generate shorter crRNAs. Each crRNA molecule consists of a unique sequence in the middle, flanked by identical sequences derived from the repeat element. The crRNA forms a hairpin-like structure, allowing for base pairing with foreign genetic material. CrRNAs assemble with one or more proteins to form an effector complex. This complex utilizes the crRNA sequence to base pair with foreign genetic material, leading to its degradation. The precise mechanism of action varies among different CRISPR systems.
There is not a single, universal CRISPR system. A large family of related CRISPR systems exists, exhibiting variations in enzyme composition and mechanisms. Researchers continue to investigate the intricate mechanisms and components involved in CRISPR-mediated immunity. Understanding these systems provides insights into microbial defense strategies and potential applications in biotechnology and medicine.
CRISPR-associated genes (cast genes) show considerable diversity in terms of their color and number of arrows. Sequence-level homology is challenging to establish due to rapid evolution. Organisms often possess multiple CRISPR loci, each with its unique type. The rapid exchange of these loci through horizontal transfer drives the evolution of CRISPR systems.
CRISPR Target DNA Requirements
CRISPR systems require a short sequence motif, known as the PAM (protospacer adjacent motif), adjacent to the sequence recognized by the CRISPR RNA. In the system being studied, the PAM motif consists of just two base pairs on the wild type strata GG. Mutations in either or both of the Gs of the PAM motif abolish cleavage and binding of the CRISPR RNA to the target DNA. Plasmids with a wild-type PAM sequence and targeted by the CRISPR system yield very few transformants. Mutations in the PAM motif restore transformation efficiency, indicating the importance of the PAM motif for CRISPR-mediated interference. DNA oligonucleotides with mutated PAM nucleotides show no cleavage or binding by the CRISPR RNA. This demonstrates the critical role of the PAM motif in CRISPR-mediated DNA targeting and binding.
Chimeric RNA Leads to Programmable DNA Endonuclease Guided by RNA
Researcher Martin Jinek created a single transcript, or chimeric RNA, that encompassed all the necessary functionalities for targeting and cleavage. This chimeric RNA included the part of the CRISPR RNA that binds DNA and the part of the tracer RNA that interacts with the CRISPR RNA and associates with Cas9 protein. Capping the ends of the RNAs with a GA3 tetraloop formed a chimeric transcript about 60 nucleotides in length. Chimera A, with a slightly longer extension on the three prime end, showed efficient DNA cleavage comparable to the natural sequence. Chimera B, truncated by about 10 nucleotides on the three prime end, exhibited no observable cleavage. Martin designed five chimeric RNAs to target specific sites on a plasmid encoding GFP. All five chimeric RNAs successfully cleaved the plasmid DNA, demonstrating the programmability of the system. To confirm the accuracy of cleavage, a restriction site near the targeted regions was used to verify that cleavage occurred at the predicted sites. The programmable DNA endonuclease guided by RNA offers advantages over existing genome editing methods, such as TALON proteins and zinc finger nucleases. Changing the sequence of a small RNA can retarget the system to different sites, eliminating the need for a new protein for each target. The successful development of the system was a result of collaboration between Jennifer Doudna’s lab and Emanuel Charpentier’s lab. Researchers from both labs contributed to the project, including Martin Jinek, Mickey Hauer, Rachel, Kai Hong, and Sam. The mechanism by which double-stranded DNA is unwound during cleavage is not yet fully understood. It is hypothesized that recognition of the PAM sequence triggers a conformational change in the effector complex, leading to local unwinding of the duplex.
Deep Dive into CRISPR-Cas9 Targeting Mechanisms
The CRISPR-Cas9 system only cuts DNA sequences flanked by a protospacer adjacent motif (PAM), a specific DNA sequence. This PAM recognition is crucial for the system’s ability to target specific DNA sequences. The molecular details of PAM recognition are not yet fully understood, but understanding this mechanism could open up more target sites in the system. The stability of the CRISPR-Cas9 complex on the DNA target raises questions about how turnover works and how new substrates are accessed. The thermodynamics of the complex may play a role in targeting efficiency and turnover. Different Cas9 variants from various organisms can cross-react with each other’s dual RNAs, enabling the system to target diverse DNA sequences. The ability of Cas9 variants to bind to different RNA structures provides opportunities for expanding the target range of the system. Single point mutations in the CRISPR-Cas9 system may affect cleavage activity but not necessarily binding. These mutations could result in a stable complex formation without cleavage, suggesting that all necessary components for binding are still present. Bacteriophages, viruses that infect bacteria, try to avoid having PAM sites in their genomes to escape the CRISPR-Cas9 system. Phages mutate their PAM sites to evade the CRISPR system, highlighting the effectiveness of this defense mechanism.
Jennifer Doudna’s research focuses on how small RNAs (guide RNAs) are generated in CRISPR systems. These guide RNAs assemble with proteins to destroy specific nucleic acids complementary to the CRISPR RNA. Doudna collaborated with Emanuel Charpentier, a microbiologist from Umea University in Sweden, who was studying the CRISPR locus in S. pyogenes, a human pathogen. The CRISPR locus in S. pyogenes contains a characteristic set of CRISPR repeats and four cast genes. This system has a smaller number of genes compared to other CRISPR pathways.
Cas9 is a dual RNA-guided DNA endonuclease
Jennifer Doudna and Emanuel Charpentier began collaborating to investigate the function of Cas9, a protein involved in the CRISPR system. Cas9 is a large protein that had been challenging to study due to difficulties in producing a purified version. In the CRISPR locus studied, CRISPR RNAs are generated through a unique process involving initial transcription of the CRISPR locus to form a long precursor molecule, base pairing of the precursor molecule with tracer RNAs to form short duplexes, and cleavage of the duplexes by the host ribonuclease 3 enzyme and Cas9, resulting in mature CRISPR RNAs. Cas9 was found to be required for blocking plasmid transformation in cells when the plasmids contained sequences matching CRISPR RNAs. This observation suggested that Cas9 might possess endonuclease activity capable of cutting DNA in an RNA-guided manner. Experiments revealed that Cas9 is a dual RNA-guided DNA endonuclease, requiring both a CRISPR RNA and a tracer RNA to function. Cleavage of plasmid DNA by Cas9 was only observed in the presence of both RNAs. Cas9 could cleave both circular and pre-linearized plasmid DNA, indicating that circularization or supercoiling was not necessary for cleavage. This discovery provided strong evidence supporting the hypothesis that Cas9 is an endonuclease capable of RNA-guided DNA cleavage. It paved the way for further research and development of Cas9 as a versatile gene-editing tool, revolutionizing the field of genome engineering.
Insights into the Cleavage Mechanism of the Cas9 Protein and Its Target Recognition Features
Cas9 creates double-strand breaks (DSBs) at specific target sites in DNA. These cleavage sites occur upstream of the end of the CRISPR RNA-target DNA base pairing region. The Proto Spacer Adjacent Motif (PAM) is a short DNA sequence essential for target recognition by Cas9. It is located immediately downstream of the CRISPR RNA-target DNA base pairing region. A tracer RNA can base pair with a sequence in the CRISPR RNA adjacent to the target-binding region. This interaction forms a complex between the two RNAs, the two DNA strands, and the Cas9 protein. Cas9 exhibits both endonuclease and 3′ to 5′ exonuclease activities. It rapidly cleaves both DNA strands at the target site, followed by further trimming of the non-complementary strand from the 3′ end. Cas9 can cleave multiple DNA molecules per complex with CRISPR RNA. This indicates that the Cas9-RNA complex undergoes multiple rounds of target recognition and cleavage.
Cas9 Catalytic Domains and Tracer RNA
Cas9 has two known nuclease domains: the RUBC and HNH nuclease domains. Mutations in either of these domains result in nicking of supercoiled DNA instead of full cleavage. The HNH nuclease domain is essential for cleaving the complementary strand, while the Rov-C domain is essential for cleaving the non-complementary strand. Tracer RNA is essential for target binding and possibly for binding and orienting the CRISPR RNA. Without the tracer RNA, there is very little formation of any complex between Cas9 and DNA. The tracer RNA does not have any complementarity to the targeted DNA. Cas9 binds to a complex of CRISPR RNA and tracer RNA, forming a larger RNP. The CRISPR RNA sequence base pairs with one strand of the DNA sequence, leading to its cleavage by the HNH and Rov-C active sites in Cas9. Cleavage occurs three base pairs upstream of the PAM motif. Each nuclease domain cleaves one strand of the DNA.
The CRISPR-Cas system’s discovery has revolutionized genome editing, offering a powerful tool for precise genetic modifications. The meeting underscored the system’s potential applications in various fields, from medicine to agriculture. Ongoing research aims to understand the minimal requirements for CRISPR and tracer RNAs, the mechanism of PAM recognition, and the impact of chromatinization on targeting and turnover.
The Cold Spring Harbor meeting illuminated the vast potential of the CRISPR-Cas system, marking a new era in genetic research. The collaborative efforts and findings presented at the meeting not only enhanced our understanding of bacterial immune systems but also paved the way for innovative approaches in gene editing, with far-reaching implications across multiple disciplines. The CRISPR-Cas system stands as a testament to the power of scientific inquiry and collaboration in unraveling the complexities of life at its most fundamental level.
The CRISPR-Cas9 system, pioneered by Jennifer Doudna, revolutionized genome editing with its ability to target specific DNA sequences, opening up new avenues in biomedical research and gene therapy. CRISPR-Cas9's dual RNA-guided DNA cleaving mechanism enables precise genome editing and has profound implications in the fields of medicine and genetics....
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....
Jennifer Doudna's extensive research on CRISPR-Cas systems revolutionized genome editing, enabling precise DNA manipulation for gene editing and correction of genetic defects. Cas9, a crucial protein in CRISPR-Cas systems, functions using two RNA molecules for DNA targeting and can cause double-stranded breaks in DNA....
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-Cas9 revolutionizes genome editing and expands its applications. CRISPR-Cas9's versatility raises ethical and societal concerns, necessitating responsible management of its technology....
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....