Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.

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Mechanism of R-loop formation and conformational activation of Cas9

10.1101/2021.09.16.460614 ◽

2021 ◽

Author(s):

Martin Pacesa ◽

Martin Jinek

Keyword(s):

Dna Cleavage ◽

Structural Basis ◽

Seed Region ◽

Loop Formation ◽

Base Pairs ◽

Guide Rna ◽

Target Strand ◽

Target Dna ◽

Dna Strand ◽

Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

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Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

Nature Communications ◽

10.1038/s41467-021-23876-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bo Zhang ◽

Diyin Luo ◽

Yu Li ◽

Vanja Perčulija ◽

Jing Chen ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Binary Complex ◽

Loop Formation ◽

Dna Duplex ◽

Target Dna ◽

Before And After ◽

Dna Strand ◽

Type V ◽

Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

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Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

10.1101/353748 ◽

2018 ◽

Cited By ~ 2

Author(s):

Daan C. Swarts ◽

Martin Jinek

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Cleavage Product ◽

List Type ◽

Francisella Novicida ◽

Ssdna Binding ◽

Double Stranded Dna ◽

Target Dna ◽

Dna Strands ◽

Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

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Structure of the Cpf1 endonuclease R-Loop complex after target DNA cleavage

10.1101/122648 ◽

2017 ◽

Cited By ~ 1

Author(s):

Stefano Stella ◽

Pablo Alcón ◽

Guillermo Montoya

Keyword(s):

Dna Cleavage ◽

Longitudinal Axis ◽

Modification System ◽

Francisella Novicida ◽

Helix Loop Helix ◽

Phosphate Backbone ◽

Target Dna ◽

Catalytically Active ◽

Dna Strand ◽

Type V

AbstractCpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45° angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 Å away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a “rail shape” and “flap-on” conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a “septum” that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNAase sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system2-4.

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Making the cut(s): how Cas12a cleaves target and non-target DNA

Biochemical Society Transactions ◽

10.1042/bst20190564 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1499-1510 ◽

Cited By ~ 5

Author(s):

Daan C. Swarts

Keyword(s):

Conformational Changes ◽

Dna Sequences ◽

Dna Cleavage ◽

Transcriptional Control ◽

Molecular Mechanisms ◽

Nucleic Acid Detection ◽

Target Dna ◽

Activated State ◽

Dna Strand ◽

Cis And Trans

Abstract CRISPR–Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a–RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR–Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

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Rational design of a split-Cas9 enzyme complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501698112 ◽

2015 ◽

Vol 112 (10) ◽

pp. 2984-2989 ◽

Cited By ~ 175

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.

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Decision letter: Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

10.7554/elife.46500.023 ◽

2019 ◽

Keyword(s):

Streptococcus Pyogenes ◽

Nuclease Domain ◽

Bona Fide

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Real-time observation of flexible domain movements in Cas9

10.1101/122069 ◽

2017 ◽

Cited By ~ 3

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.

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Structural basis for Cas9 off-target activity

10.1101/2021.11.18.469088 ◽

2021 ◽

Author(s):

Martin Pacesa ◽

Chun-Han Lin ◽

Antoine Clery ◽

Katja Bargsten ◽

Matthew J. Irby ◽

...

Keyword(s):

Rational Design ◽

Target Prediction ◽

Structural Basis ◽

Guide Rna ◽

Guide Rnas ◽

Target Dna ◽

Bona Fide ◽

Dna Specificity ◽

Target Binding ◽

Target Activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non- canonical base pairing interactions and preservation of base stacking within the guide-off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

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Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

Life Science Alliance ◽

10.26508/lsa.202101078 ◽

2022 ◽

Vol 5 (4) ◽

pp. e202101078

Author(s):

Tunahan Ergünay ◽

Özgecan Ayhan ◽

Arda B Celen ◽

Panagiota Georgiadou ◽

Emre Pekbilir ◽

...

Keyword(s):

Dna Binding ◽

Regulatory Mechanisms ◽

Clinical Implementation ◽

Post Translational Modifications ◽

Guide Rna ◽

Nuclease Domain ◽

Target Dna ◽

Unwanted Consequence ◽

Positively Charged ◽

Pharmacologic Inhibition

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

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