scholarly journals Structural basis for mismatch surveillance by CRISPR/Cas9

2021 ◽  
Author(s):  
Jack PK Bravo ◽  
Mu-Sen Liu ◽  
Ryan S McCool ◽  
Kyungseok Jung ◽  
Kenneth A Johnson ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Structural Basis ◽  
Guide Rna ◽  
Flexible Loop ◽  
Target Strand ◽  
Target Dna ◽  
Conformational Rearrangements ◽  
Structural Mechanisms

The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016), the underlying molecular mechanisms by which Cas9 rejects or accepts mismatches are poorly understood (Kim et al., 2019; Liu et al., 2020; Slaymaker and Gaudelli, 2021). Here, we used kinetic analysis to guide cryo-EM structure determination of Cas9 at different stages of mismatch surveillance. We observe a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS), that occurs in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. We observe that highly tolerated PAM- distal mismatches achieve this kinked conformation through stabilization of a distorted duplex conformation via a flexible loop in the RuvC domain. Our results provide molecular insights into the underlying structural mechanisms that may facilitate off- target cleavage by Cas9 and provides a molecular blueprint for the design of next- generation high fidelity Cas9 variants that selectively reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

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.

scholarly journals Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

2021 ◽  
Vol 118 (49) ◽  
pp. e2113747118
Author(s):  
Heyjin Son ◽  
Jaeil Park ◽  
Injoo Hwang ◽  
Youngri Jung ◽  
Sangsu Bae ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Resonance Energy ◽  
Distal Region ◽  
Catalytic Site ◽  
Nucleic Acid Detection ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Conformational Rearrangements

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease

2020 ◽  
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

ABSTRACTCas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

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

2019 ◽  
Vol 47 (5) ◽  
pp. 1499-1510 ◽  
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.

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 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 Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

2021 ◽  
Author(s):  
Renjian Xiao ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Zhuang Li ◽  
Clinton Gabel ◽  
...  
Keyword(s):  
Dna Recognition ◽  
Structural Basis ◽  
Structural Elements ◽  
Dna Transposition ◽  
Guide Rna ◽  
Promising Tool ◽  
Target Dna ◽  
Dna Insertion ◽  
Type V

The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryo-EM structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT PAM sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing.

scholarly journals Structural basis for Cas9 off-target activity

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.

scholarly journals Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

2021 ◽  
Vol 49 (7) ◽  
pp. 4120-4128
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

Abstract Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

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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