Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex.

The type III-E CRISPR-Cas effector Cas7-11, with dual RNase activities for precursor CRISPR RNA (pre-crRNA) processing and crRNA-guided target RNA cleavage, is a new platform for bacterial and mammalian RNA targeting. We report the 2.5-Å resolution cryoelectron microscopy structure of Cas7-11 in complex with a crRNA and its target RNA. Cas7-11 adopts a modular architecture comprising seven domains (Cas7.1-Cas7.4, Cas11, INS, and CTE) and four interdomain linkers. The crRNA 5' tag is recognized and processed by Cas7.1, whereas the crRNA spacer hybridizes with the target RNA. Consistent with our biochemical data, the catalytic residues for programmable cleavage in Cas7.2 and Cas7.3 neighbor the scissile phosphates before the flipped-out fourth and tenth nucleotides in the target RNA, respectively. Using structural insights, we rationally engineered a compact Cas7-11 variant (Cas7-11S) for single-vector AAV packaging for transcript knockdown in human cells, enabling in vivo Cas7-11 applications.

Type III CRISPR-Cas: beyond the Cas10 effector complex.

Type III CRISPR-Cas loci encode some of the most abundant, yet complex, immune systems of prokaryotes. They are composed of a Cas10 complex that uses an RNA guide to recognize transcripts from bacteriophage and plasmid invaders. Target recognition triggers three activities within this complex: ssDNA degradation, synthesis of cyclic oligoadenylates (cOA) that act as second messengers to activate CARF-domain effectors, and cleavage of target RNA. This review covers recent research in type III CRISPR-Cas systems that looked beyond the activity of the canonical Cas10 complexes towards: (i) ancillary nucleases and understanding how they provide defense by sensing cOA molecules; (ii) ring nucleases and their role in regulating cOA production; and (iii) CRISPR-associated proteases, including the function of the Craspase complex in a transcriptional response to phage infection.

Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems.

RNA-guided protease activity was recently discovered in the type III-E CRISPR-Cas systems (Craspase), providing a novel platform for engineering a protein probe instead of the commonly used nucleic acid probe in nucleic acid detection assays. Here, by adapting a fluorescence readout technique using the affinity- and fluorescent protein dual-tagged Csx30 protein substrate, we have established an assay monitoring Csx30 cleavage by target ssRNA-activated Craspase. Four Craspase-based nucleic acid detection systems for genes from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), norovirus, and the influenza virus (IFV) were reconstituted with demonstrated specificity. The assay could reliably detect target ssRNAs at concentrations down to 25 pM, which could be further improved approximately 15 000-fold (ca. 2 fM) by incorporating a recombinase polymerase isothermal preamplification step. Importantly, the species-specific substrate cleavage specificity of Craspase enabled multiplexed diagnosis, as demonstrated by the reconstituted composite systems for simultaneous detection of two genes from the same virus (SARS-CoV-2, spike and nsp12) or two types of viruses (SARS-CoV-2 and IFV). The assay could be further expanded by diversifying the fluorescent tags in the substrate and including Craspase systems from various species, thus potentially providing an easily adaptable platform for clinical diagnosis.

Novel molecular aspects of the CRISPR backbone protein 'Cas7' from cyanobacteria.

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.

Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.

Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family.

Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of Thermofilum pendens Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. T. pendens Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on T. pendens Csc2. Thus our analysis of the key structural and functional features of T. pendens Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish.

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

Structure and RNA-binding properties of the type III-A CRISPR-associated protein Csm3.

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.

Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum.

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex. In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.

[Advances in the RNA-targeting CRISPR-Cas systems].

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.

Assembly of Cas7 subunits of Leptospira on the mature crRNA of CRISPR-Cas I-B is modulated by divalent ions.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (Kd = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA.

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.

Ribonucleic acid or RNA is an important molecule involved in making proteins, transmitting diseases, offering immunity in form of vaccines, and also degrading itself. Programmed RNA degradation is a common method used by bacteria to protect themselves from invading viruses. Bacteria acquire viral genetic materials during infections, which are then converted into RNA fragments, or guide RNA. The guide RNAs both locate and recruit enzymes to help destroy the infectious RNA. These programmable RNA degradation machineries can be repurposed for biotechnology applications to help regulate gene expression or to minimize the effect of viral infections. Similar machineries, like the CRISPR/Cas9 gene editing tool, act like genetic scissors, allowing researchers to make precise modifications to DNA to study and alter the role of genes in the cell. Like in bacteria, the CRISPR system uses fragments of RNA from viruses as a guide to identify matching targets and create breakages in the genetic material. Recently, researchers discovered Cas7-11, which is used to break sections of RNA in viruses. To better understand how Cas7-11 works, Goswami et al. studied its three-dimensional structure. Detailed views of each segment of the protein, together with biochemical studies of the protein's activity, helped to identify their respective roles. The structural information also highlighted three regions involved in snipping RNA and revealed how they drive this process. This analysis showed that a short segment of Cas7-11 alone is sufficient to prepare and bind the guide RNA fragments. These findings add to the understanding of how Cas7-11 prepares its guide and creates breakages in RNA. It has a similar structure to a previously known assembly of proteins that also breaks down RNA, providing insight into its evolution. The detailed analysis of how Cas7-11 works also demonstrates the possibility of engineering it as a laboratory tool to remove specific RNA sequences in cells.

Positioning Diverse Type IV Structures and Functions Within Class 1 CRISPR-Cas Systems.

Type IV CRISPR systems encode CRISPR associated (Cas)-like proteins that combine with small RNAs to form multi-subunit ribonucleoprotein complexes. However, the lack of Cas nucleases, integrases, and other genetic features commonly observed in most CRISPR systems has made it difficult to predict type IV mechanisms of action and biological function. Here we summarize recent bioinformatic and experimental advancements that collectively provide the first glimpses into the function of specific type IV subtypes. We also provide a bioinformatic and structural analysis of type IV-specific proteins within the context of multi-subunit (class 1) CRISPR systems, informing future studies aimed at elucidating the function of these cryptic systems.

Predicted highly derived class 1 CRISPR-Cas system in Haloarchaea containing diverged Cas5 and Cas7 homologs but no CRISPR array.

Screening of genomic and metagenomic databases for new variants of CRISPR-Cas systems increasingly results in the discovery of derived variants that do not seem to possess the interference capacity and are implicated in functions distinct from adaptive immunity. We describe an extremely derived putative class 1 CRISPR-Cas system that is present in many Halobacteria and consists of distant homologs of the Cas5 and Cas7 protein along with an uncharacterized conserved protein and various nucleases. We hypothesize that, although this system lacks typical CRISPR effectors or a CRISPR array, it functions as a RNA-dependent defense mechanism that, unlike other derived CRISPR-Cas, utilizes alternative nucleases to cleave invader genomes.

[Effect of electroacupuncture on expressions of Gas7 and NGF in arcular nucleus of rats with focal cerebral ischemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expressions of growth arrest-specific protein 7 (Gas7) and nerve growth factor (NGF) in arcuate nucleus (ARC) of rats with focal cerebral ischemia and explore the potential action mechanism of EA in treatment of focal cerebral ischemia. METHODS: A total of 50 SD rats were randomized into 4 groups, named a normal group (n =12), a sham-operation group (n =12), a model group (n =14) and an EA group (n =12). In the model group and the EA group, the thread embolization method was adopted to duplicate the model of the right middle cerebral arterial embolism. In the sham-operation group, the skin of the neck was opened and sutured without any other intervention. In the EA group, EA was applied to "Baihui" (CV 20) and "Zusanli" (ST 36) on the left side, once a day, 30 min each time, consecutively for 21 days, while there was no any intervention in the normal group, the sham-operation group and the model group. Using the immunohistochemistry (IHC) method and Western blot method, the expressions of Gas7 and NFG of ARC on the ischemic side were determined. Using Nissle staining, the morphological changes in ARC neurons were observed. RESULTS: The results of Nissle staining showed that there was no significant change in the morphology of ARC neurons in the normal group and the sham-operation group. In the model group, the volume of neuron cells was atrophied obviously and the cells were arranged irregularly. In the EA group, the morphology of ARC neuron was similar to the normal group. The results of IHC and Western blot indicated that the expressions of immunoreactive neurons and protein of Gas7 and NGF in ARC of the rats in the model group were increased obviously as compared with the normal group and the sham-operation group and the expressions in the EA group were further enhanced as compared with the model group (all P<0.05). CONCLUSION: Gas7 and NGF may be participated in the compensatory process of partial protection of the body in the patients with focal cerebral ischemia. EA up-regulates the expressions of Gas7 and NGF in ARC, which may be one of the neuroprotective mechanisms of EA in treatment of cerebral ischemia.

Advances in the RNA-targeting CRISPR-Cas systems / 生物工程学报

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.

Effect of electroacupuncture on expressions of Gas7 and NGF in arcular nucleus of rats with focal cerebral ischemia / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) on the expressions of growth arrest-specific protein 7 (Gas7) and nerve growth factor (NGF) in arcuate nucleus (ARC) of rats with focal cerebral ischemia and explore the potential action mechanism of EA in treatment of focal cerebral ischemia.@*METHODS@#A total of 50 SD rats were randomized into 4 groups, named a normal group ( =12), a sham-operation group ( =12), a model group ( =14) and an EA group ( =12). In the model group and the EA group, the thread embolization method was adopted to duplicate the model of the right middle cerebral arterial embolism. In the sham-operation group, the skin of the neck was opened and sutured without any other intervention. In the EA group, EA was applied to "Baihui" (CV 20) and "Zusanli" (ST 36) on the left side, once a day, 30 min each time, consecutively for 21 days, while there was no any intervention in the normal group, the sham-operation group and the model group. Using the immunohistochemistry (IHC) method and Western blot method, the expressions of Gas7 and NFG of ARC on the ischemic side were determined. Using Nissle staining, the morphological changes in ARC neurons were observed.@*RESULTS@#The results of Nissle staining showed that there was no significant change in the morphology of ARC neurons in the normal group and the sham-operation group. In the model group, the volume of neuron cells was atrophied obviously and the cells were arranged irregularly. In the EA group, the morphology of ARC neuron was similar to the normal group. The results of IHC and Western blot indicated that the expressions of immunoreactive neurons and protein of Gas7 and NGF in ARC of the rats in the model group were increased obviously as compared with the normal group and the sham-operation group and the expressions in the EA group were further enhanced as compared with the model group (all <0.05).@*CONCLUSION@#Gas7 and NGF may be participated in the compensatory process of partial protection of the body in the patients with focal cerebral ischemia. EA up-regulates the expressions of Gas7 and NGF in ARC, which may be one of the neuroprotective mechanisms of EA in treatment of cerebral ischemia.