Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5′ tag of crRNA, the 3′ anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
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•Crystal structure of the type III-A CRISPR-RNA-guided surveillance complex•Cryo-EM structures of cognate and non-cognate target RNA-bound Csm complexes•Complementary and non-complementary 3′ anti-tag binds at two distinct sites•Target RNA binding allosterically activates ssDNA cleavage and cOA synthesis
Structures of the type III-A CRISPR-Csm complex in the presence of target RNAs reveal the molecular basis for a unique mode of self/non-self discrimination.
Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and ...archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5' and 3' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long β-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each β-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
CTCF, a conserved 3D genome architecture protein, determines proper genome-wide chromatin looping interactions through directional binding to specific sequence elements of four modules within ...numerous CTCF-binding sites （CBSs） by its 11 zinc fingers （ZFs）. Here, we report four crystal structures of human CTCF in complex with CBSs of the protocadherin （Pcdh） clusters. We show that directional CTCF binding to cognate CBSs of the Pcdh enhancers and promoters is achieved through inserting its ZF3, ZFs 4-7, and ZFs 9-11 into the major groove along CBSs, result- ing in a sequence-specific recognition of module 4, modules 3 and 2, and module 1, respectively; and ZF8 serves as a spacer element for variable distances between modules 1 and 2. In addition, the base contact with the asymmetric ＂A＂ in the central position of modules 2-3, is essential for directional recognition of the CBSs with symmetric core sequences but lacking module 1. Furthermore, CTCF tolerates base changes at specific positions within the degen- erated CBS sequences, permitting genome-wide CTCF binding to a diverse range of CBSs. Together, these complex structures provide important insights into the molecular mechanisms for the directionality, diversity, flexibility, dynamics, and conservation of multivalent CTCF binding to its cognate sites across the entire human genome.
Abstract
To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically ...competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.
AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two ...activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr's C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15's inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.
We report on crystal structures of ternary Thermus thermophilus Argonaute (Tt Ago) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of ...cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg ²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for Tt Ago-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.
Dear Editor,
CRISPR/Cas systems are widespread RNA-mediated prokaryotic adaptive immune systems providing protection against invading nucleic acids . However, throughout evolution, this host defense ...system has not resulted in the eradication of phages, suggesting that phages have evolved counter strategies to thrive within bacteria despite these mechanisms. Thus, both bacterial CRISPR system and phage anti-CRISPR system are part of a continuing evolutionary battle between bacterial host and their bacteriophage invaders.
Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a ...is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
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•Structures of L. buccalis (Lbu) Cas13a in pre-target and target-bound states•Cas13a and crRNA undergo a significant conformational change upon target RNA binding•The formation of the guide-target RNA duplex activates HEPN catalytic site of Cas13a•Activated Cas13a cleaves exposed regions of ssRNA in solution
Structural analysis of CRISPR-Cas13a (C2c2) reveals how target RNA binding induces a conformational change to activate non-specific RNA degradation.
Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of ...Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3′ overhang segments. The PAM-complementary sequence in the 3′ overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.
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•The dual-forked protospacer is integrated via a cut-and-paste mechanism•Architecture of Cas1-Cas2 predetermines length of newly acquired spacer•Cas1a recognizes PAM-complementary sequence via sequence-specific interactions•Cas1-Cas2 undergoes a conformational change upon protospacer DNA binding
Cas1 and Cas2 select an invading DNA sequence, termed protospacer, for insertion into the CRISPR locus of the host cell. The structure of the Cas1-Cas2-protospacer DNA complex reveals the dual-forked nature of the protospacer, explains how the protospacer is selected, and identifies how protospacer length is predetermined.
C2c1 is a type V-B CRISPR-Cas system dual-RNA-guided DNA endonuclease. Here, we report the crystal structure of Alicyclobacillus acidoterrestris C2c1 in complex with a chimeric single-molecule guide ...RNA (sgRNA). AacC2c1 exhibits a bi-lobed architecture consisting of a REC and NUC lobe. The sgRNA scaffold forms a tetra-helical structure, distinct from previous predictions. The crRNA is located in the central channel of C2c1, and the tracrRNA resides in an external surface groove. Although AacC2c1 lacks a PAM-interacting domain, our analysis revealed that the PAM duplex has a similar binding position found in Cpf1. Importantly, C2c1-sgRNA system is highly sensitive to single-nucleotide mismatches between guide RNA and target DNA. The resulting reduction in off-target cleavage renders C2c1 a valuable addition to the current arsenal of genome-editing tools. Together, our findings indicate that sgRNA assembly is achieved through a mechanism distinct from that reported previously for Cas9 or Cpf1 endonucleases.
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•Crystal structure of Alicyclobacillus acidoterrestris C2c1-sgRNA complex•Mechanistic insights into dual-RNA-guided, C2c1-catalyzed, staggered dsDNA breaks•High mismatch sensitivity of C2c1 reduces off-target cleavage•Join 2/4-truncated sgRNA-guided DNA cleavage similar to that of wild-type sgRNA
Liu et al. report the structure of C2c1-a type V-B CRISPR-Cas endonuclease, in complex with a chimeric single guide RNA, providing insights into the high specificity of DNA cleavage by C2c1. C2c1’s low off-target rate makes it a valuable addition to the current arsenal of gene-editing tools.
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