scholarly journals Real-time observation of flexible domain movements in Cas9

2017 ◽  
Author(s):  
Saki Osuka ◽  
Kazushi Isomura ◽  
Shohei Kajimoto ◽  
Tomotaka Komori ◽  
Hiroshi Nishimasu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Potential Role ◽  
Domain Architecture ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Dna ◽  
Time Observation ◽  
Real Time Observation

ABSTRACTThe CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

scholarly journals Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

2016 ◽  
Author(s):  
Digvijay Singh ◽  
Samuel H. Sternberg ◽  
Jingyi Fei ◽  
Jennifer A. Doudna ◽  
Taekjip Ha
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Dissociation Rate ◽  
Stable Complex ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Protospacer Adjacent Motif ◽  
Time Observation

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome engineering applications, but how mismatches influence target recognition and rejection kinetics is not well understood. We used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence, but the dissociation rate greatly increases from < 0.006 s-1 to > 2 s-1 upon introduction of mismatches proximal to the protospacer adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 12 base pairs in length, which prevent DNA cleavage, still allow the formation of a stable complex (off-rate < 0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome editing thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

scholarly journals A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

2017 ◽  
Author(s):  
Yavuz S. Dagdas ◽  
Janice S. Chen ◽  
Samuel H. Sternberg ◽  
Jennifer A. Doudna ◽  
Ahmet Yildiz
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Divalent Cations ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Sites ◽  
Multiple Conformations ◽  
Dna Binding And Cleavage

AbstractThe Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

scholarly journals Real-Time Observation of DNA Recognition by the RNA-Guided Endonuclease Cas9 using Single-Molecule FRET

2015 ◽  
Vol 108 (2) ◽  
pp. 26a
Author(s):  
Digvijay Singh ◽  
Samuel H. Sternberg ◽  
Jingyi Fei ◽  
Jennifer A. Doudna ◽  
Taekjip Ha
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Dna Recognition ◽  
Single Molecule Fret ◽  
Time Observation ◽  
Real Time Observation

scholarly journals Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

2018 ◽  
Vol 115 (21) ◽  
pp. 5444-5449 ◽  
Author(s):  
Digvijay Singh ◽  
John Mallon ◽  
Anustup Poddar ◽  
Yanbo Wang ◽  
Ramreddy Tippana ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Fluorescence Analysis ◽  
Dna Interaction ◽  
Target Sequence ◽  
Solution Ph ◽  
Guide Rna ◽  
Product Release ◽  
Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

scholarly journals Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

2017 ◽  
Author(s):  
Digvijay Singh ◽  
John Mallon ◽  
Anustup Poddar ◽  
Yanbo Wang ◽  
Ramreddy Tippana ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Fluorescence Analysis ◽  
Dna Interaction ◽  
Target Sequence ◽  
Solution Ph ◽  
Guide Rna ◽  
Product Release ◽  
Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9, they both bind any DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence directionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ~16 bp for cleavage, Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions and 5’ guanine in guide-RNA differentially affected different Cpf1 orthologues. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

scholarly journals Real-Time Observation of Multiple-Protein Complex Formation with Single-Molecule FRET

2013 ◽  
Vol 135 (28) ◽  
pp. 10254-10257 ◽  
Author(s):  
Woori Bae ◽  
Mal-Gi Choi ◽  
Changbong Hyeon ◽  
Yeon-Kyun Shin ◽  
Tae-Young Yoon
Keyword(s):  
Complex Formation ◽  
Real Time ◽  
Single Molecule ◽  
Protein Complex ◽  
Single Molecule Fret ◽  
Protein Complex Formation ◽  
Multiple Protein ◽  
Time Observation ◽  
Real Time Observation

scholarly journals Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

2020 ◽  
Vol 6 (19) ◽  
pp. eaay9948
Author(s):  
Fei Wang ◽  
Yaya Hao ◽  
Qian Li ◽  
Jiang Li ◽  
Honglu Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Target Dna

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

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