Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.     

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.     

Conservation and Variability in the Structure and Function of the Cas5d Endoribonuclease in the CRISPR-Mediated Microbial Immune System

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR–Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event.     

Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca²⁺ was located at strategic positions in the oligomerization interface. We further showed that removal of Ca²⁺ ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca²⁺ ions.     

Crystal structure of Cas1 from Archaeoglobus fulgidus and characterization of its nucleolytic activity

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins are involved in bacterial acquired immunity against incoming hazardous genetic materials. Cas1 is ubiquitous in CRISPR-containing microorganisms and supposed to recognize and cleave a foreign nucleic acid, and integrate the cleaved fragment into host genome using a yet unidentified mechanism. However, all the reported Cas1s did not show the nucleolytic activity, which makes its role still obscure. The elucidated crystal structure of Cas1 from Archaeoglobus fulgidus (AfCas1) shows a butterfly-like dimeric structure. The Asp out of three confirmed nucleolytic residues of Glu, His, and Asp in other Cas1s is replaced with Glu in AfCas1. Further, insertion of five residues into one of two loops, which are close to the catalytic center of and disordered in other Cas1 structures, partially covers the active site of AfCas1. Nonetheless, in vitro assays show that its nucleic acid-binding activity was not impaired against the tested single-stranded (ss) DNA, various forms of double-stranded (ds) DNA, or ssRNA with a hydrolyzing activity against ssRNA and dsDNA in a metal ion-dependent way. These results support the proposed Cas1’s function at the early step of this bacterial immune system.     

Identification,structural, and biochemical characterization of a group of large Csn2 proteins involved in CRISPR‐mediated bacterial immunity

Many prokaryotic organisms acquire immunity against foreign genetic material by incorporating a short segment of foreign DNA called spacer into chromosomal loci, termed clustered regularly interspaced short palindromic repeats (CRISPRs). The encoded RNAs are processed into small fragments that guide the silencing of the invading genetic elements. The CRISPR‐associated (Cas) proteins are the main executioners of these processes. Herein, we report the crystal structure of Stu0660 of Streptococcus thermophilus, a Cas protein involved in the acquisition of new spacers. By homotetramerization, Stu0660 forms a central channel which is decorated with basic amino acids and binds linear double‐stranded DNA (dsDNA), but not circular dsDNA. Despite undetectably low sequence similarity, two N‐terminal domains of Stu0660 are similar to the entire structure of an Enterococcus faecalis Csn2 protein, which also forms a homotetramer and binds dsDNA. Thus, this work identifies a previously unknown group of Stu0660‐like Csn2 proteins (～350 residues), which are larger than the known canonical Csn2 proteins (～220 residues) by containing an extra C‐terminal domain. The commonly present central channel in the two subgroups appears as a design to selectively interact with linear dsDNA. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Structural and dynamic insights into the HNH nuclease of divergent Cas9 species

CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.     

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR–Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)₃₃. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.     

Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.     

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.     

Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants

CRISPR/Cas9 technology has been used to edit genomes in a variety of organisms. Using the GP72 gene as a target sequence, we tested two distinct approaches to generate Trypanosoma cruzi knockout mutants using the Cas9 nuclease and in vitro transcribed single guide RNA. Highly efficient rates of disruption of GP72 were achieved either by transfecting parasites stably expressing Streptococcus pyogenes Cas9 with single guide RNA or by transfecting wild type parasites with recombinant Staphylococcus aureus Cas9 previously associated with single guide RNA. In both protocols, we used single-stranded oligonucleotides as a repair template for homologous recombination and insertion of stop codons in the target gene.     

COP9 Signalosome Subunit Csn8 Is Involved in Maintaining Proper Duration of the G1 Phase

The COP9 signalosome (CSN) is a conserved protein complex known to be involved in developmental processes of eukaryotic organisms. Genetic disruption of a CSN gene causes arrest during early embryonic development in mice. The Csn8 subunit is the smallest and the least conserved subunit, being absent from the CSN complex of several fungal species. Nevertheless, Csn8 is an integral component of the CSN complex in higher eukaryotes, where it is essential for life. By characterizing the mouse embryonic fibroblasts (MEFs) that express Csn8 at a low level, we found that Csn8 plays an important role in maintaining the proper duration of the G₁ phase of the cell cycle. A decreased level of Csn8, either in Csn8 hypomorphic MEFs or following siRNA-mediated knockdown in HeLa cells, accelerated cell growth rate. Csn8 hypomorphic MEFs exhibited a shortened G₁ duration and affected expression of G₁ regulators. In contrast to Csn8, down-regulation of Csn5 impaired cell proliferation. Csn5 proteins were found both as a component of the CSN complex and outside of CSN (Csn5-f), and the amount of Csn5-f relative to CSN was increased in the Csn8 hypomorphic cells. We conclude that CSN harbors both positive and negative regulators of the cell cycle and therefore is poised to influence the fate of a cell at the crossroad of cell division, differentiation, and senescence.     

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.     

Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.     

Diversity of family GH46 chitosanases in Kitasatospora setae KM-6054

The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid. Kinetic parameters were also determined. These tests unveiled the biochemical diversity among these chitosanases and the peculiarity of Csn3 compared with the other three enzymes. The observed biochemical diversity is discussed based on structural 3D models and sequence alignment. This is a first study of all the GH46 chitosanases produced by a single microbial strain.

     

Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco, sorghum and rice

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13 ^−/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.