Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.

Method to generate Holliday junction recombination intermediates via RecA-mediated four-strand exchange.

DNA molecules that contain single Holliday junctions have served as model substrates to investigate the pathway in which homologous recombination intermediates are processed. However, the preparation of DNA containing Holliday junctions in high yield remains a challenge. In this work, we used a nicking endonuclease to generate gapped DNA, from which α-structured DNA or figure-8 DNA were created via RecA-mediated reactions. The resulting DNA molecules were found to serve as good substrates for Holliday junction resolvases. The simplified method negates the requirement for radioactive labelling of DNA, making the generation of Holliday junction DNA more accessible to non-experts.

Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.

Biochemical and structural characterization of the Holliday junction resolvase RuvC from Pseudomonas aeruginosa.

The Holliday junction, a four-way DNA structure, is an important intermediate of homologous recombination. Proper Holliday junction resolution is critical to complete the recombination process. In most bacterial cells, the Holliday junction cleavage is mainly performed by a specific endonuclease RuvC. Here, we describe the biochemical properties and the crystal structure of RuvC from an opportunistic pathogen, Pseudomonas aeruginosa (PaRuvC). PaRuvC specifically binds to the Holliday junction DNA and preferentially cleaves it at the consensus 5'-TTC-3'. PaRuvC uses Mg2+ as the preferred divalent metal cofactor for Holliday junction cleavage and its optimum pH is 8.0-9.0. Elevated temperatures (37-60 °C) boost the catalytic activity, but temperatures higher than 53 °C reduce the protein stability. The crystal structure of PaRuvC determined at 2.4 Å and mutagenesis analysis reveal key residues involved in the dimer formation, substrate binding and catalysis. Our results are expected to provide useful information to combat antibiotic resistance of Pseudomonas aeruginosa by targeting its homologous recombination system.

Resolution of single and double Holliday junction recombination intermediates by GEN1.

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5'-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase, Escherichia coli RuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5'-A/TTTG/C-3'.

Type II and type V CRISPR effector nucleases from a structural biologist's perspective.

The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are "universal" DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5'- or 3'-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases.

Classical and novel properties of Holliday junction resolvase SynRuvC from Synechocystis sp. PCC6803.

Cyanobacteria, which have a photoautotrophic lifestyle, are threatened by ultraviolet solar rays and the reactive oxygen species generated during photosynthesis. They can adapt to environmental conditions primarily because of their DNA damage response and repair mechanisms, notably an efficient homologous recombination repair system. However, research on double-strand break (DSB) repair pathways, including the Holliday junction (HJ) resolution process, in Synechocystis sp. PCC6803 is limited. Here, we report that SynRuvC from cyanobacteria Synechocystis sp. PCC6803 has classical HJ resolution activity. We investigated the structural specificity, sequence preference, and biochemical properties of SynRuvC. SynRuvC strongly preferred Mn2+ as a cofactor, and its cleavage site predominantly resides within the 5'-TG↓(G/A)-3' sequence. Interestingly, novel flap endonuclease and replication fork intermediate cleavage activities of SynRuvC were also determined, which distinguish it from other reported RuvCs. To explore the effect of SynRuvC on cell viability, we constructed a knockdown mutant and an overexpression strain of Synechocystis sp. PCC6803 (synruvCKD and synruvCOE) and assessed their survival under a variety of conditions. Knockdown of synruvC increased the sensitivity of cells to MMS, HU, and H2O2. The findings suggest that a novel RuvC family HJ resolvase SynRuvC is important in a variety of DNA repair processes and stress resistance in Synechocystis sp. PCC6803.

Structural and Functional Characterization of the Holliday Junction Resolvase RuvC from Deinococcus radiodurans.

Holliday junctions (HJs) are four-way DNA structures, which are an important intermediate in the process of homologous recombination. In most bacteria, HJs are cleaved by specific nucleases called RuvC resolvases at the end of homologous recombination. Deinococcus radiodurans is an extraordinary radiation-resistant bacterium and is known as an ideal model organism for elucidating DNA repair processes. Here, we described the biochemical properties and the crystal structure of RuvC from D. radiodurans (DrRuvC). DrRuvC exhibited an RNase H fold that belonged to the retroviral integrase family. Among many DNA substrates, DrRuvC specifically bound to HJ DNA and cleaved it. In particular, Mn2+ was the preferred bivalent metal co-factor for HJ cleavage, whereas high concentrations of Mg2+ inhibited the binding of DrRuvC to HJ. In addition, DrRuvC was crystallized and the crystals diffracted to 1.6 Å. The crystal structure of DrRuvC revealed essential amino acid sites for cleavage and binding activities, indicating that DrRuvC was a typical resolvase with a characteristic choice for metal co-factor.

Biochemical and Structural Study of RuvC and YqgF from Deinococcus radiodurans.

Deinococcus radiodurans possesses robust DNA damage response and repair abilities, and this is mainly due to its efficient homologous recombination repair system, which incorporates an uncharacterized Holliday junction (HJ) resolution process. D. radiodurans encodes two putative HJ resolvase (HJR) homologs: RuvC (DrRuvC) and YqgF (DrYqgF). Here, both DrRuvC and DrYqgF were identified as essential proteins for the survival of D. radiodurans. The crystal structures and the biochemical properties of DrRuvC and DrYqgF were also studied. DrRuvC crystallized as a homodimer, while DrYqgF crystallized as a monomer. DrRuvC could preferentially cleave HJ at the consensus 5'-(G/C)TC↓(G/C)-3' sequence and could prefer using Mn2+ for catalysis in vitro, which would be different from the preferences of the other previously characterized RuvCs. On the other hand, DrYqgF was identified as a Mn2+-dependent RNA 5'-3' exo/endonuclease with a sequence preference for poly(A) and without any HJR activity. IMPORTANCE Deinococcus radiodurans is one of the most radioresistant bacteria in the world due to its robust DNA damage response and repair abilities, which are contributed by its efficient homologous recombination repair system. However, the late steps of homologous recombination, especially the Holliday junction (HJ) resolution process, have not yet been well-studied in D. radiodurans. We characterized the structural and biochemical features of the two putative HJ resolvases, DrRuvC and DrYqgF, in D. radiodurans. It was identified that DrRuvC and DrYqgF exhibit HJ resolvase (HJR) activity and RNA exo/endonuclease activity, respectively. Furthermore, both DrRuvC and DrYqgF digest substrates in a sequence-specific manner with a preferred sequence that is different from those of the other characterized RuvCs or YqgFs. Our findings provide new insights into the HJ resolution process and reveal a novel RNase involved in RNA metabolism in D. radiodurans.

Preparation and Resolution of Holliday Junction DNA Recombination Intermediates.

Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.