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 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 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 Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

scholarly journals A cloning-free method for CRISPR/Cas9-mediated genome editing in fission yeast

2017 ◽  
Author(s):  
Xiao-Ran Zhang ◽  
Jia-Bei He ◽  
Yi-Zheng Wang ◽  
Li-Lin Du
Keyword(s):  
Fission Yeast ◽  
Genome Editing ◽  
Dna Cleavage ◽  
Genomic Sequence ◽  
Yeast Cells ◽  
Target Sequence ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Guide Rna ◽  
Sequence Deletion

ABSTRACTThe CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.

scholarly journals Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

2020 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Seung-Hun Kang ◽  
Junho K. Hur ◽  
Hyomin Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Genetic Mutation ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Effective Manner ◽  
Target Dna

AbstractThe CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

scholarly journals Rational design of a split-Cas9 enzyme complex

2015 ◽  
Vol 112 (10) ◽  
pp. 2984-2989 ◽  
Author(s):  
Addison V. Wright ◽  
Samuel H. Sternberg ◽  
David W. Taylor ◽  
Brett T. Staahl ◽  
Jorge A. Bardales ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Rational Design ◽  
Genome Engineering ◽  
Guide Rna ◽  
Protein Architecture ◽  
Target Dna ◽  
Molecular Determinants ◽  
Versatile Tool ◽  
Bacterial Immune Systems

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

scholarly journals Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

2017 ◽  
Author(s):  
Ryan Marshall ◽  
Colin S. Maxwell ◽  
Scott P. Collins ◽  
Michelle L. Luo ◽  
Thomas Jacobsen ◽  
...  
Keyword(s):  
Protein Purification ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Gene Repression ◽  
Live Cells ◽  
Translation System ◽  
Guide Rna ◽  
E Coli ◽  
Translation Systems

ABSTRACTCRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

scholarly journals In vitro analysis of site specific nuclease selectivity by NGS

2021 ◽  
Vol 8 (4) ◽  
pp. 235-242
Author(s):  
Vincent Brondani ◽  
Keyword(s):  
Restriction Endonuclease ◽  
Genome Engineering ◽  
Direct Interaction ◽  
Target Sequence ◽  
Vitro Assay ◽  
Guide Rna ◽  
Phosphate Backbone ◽  
Target Dna ◽  
Vitro Analysis

<abstract> <p>Nucleases currently used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. The RNA guided nucleases describe today are recognizing a sequence with two distinct molecular interactions: first, like a restriction endonuclease, by direct interaction between the protein and the DNA; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report an in vitro assay to assess the cleavage specificity and the selectivity of the nucleases. The assay is designed using a plasmid encompassing the DNA target site degenerated at positions determined on structural feature. The results demonstrate that the Cpf1 RNA guided nuclease is highly specific for the target sequence, nevertheless its substrate selectivity is low compare to a restriction endonuclease.</p> </abstract>

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