RESUMEN
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based technology has revolutionized the field of biomedicine with broad applications in genome editing, therapeutics and diagnostics. While a majority of applications involve the RNA-guided site-specific DNA or RNA cleavage by CRISPR enzymes, recent successes in nucleic acid detection rely on their collateral and non-specific cleavage activated by viral DNA or RNA. Ranging in enzyme composition, the mechanism for distinguishing self- from foreign-nucleic acids, the usage of second messengers, and enzymology, the CRISPR enzymes provide a diverse set of diagnosis tools in further innovations. Structural biology plays an important role in elucidating the mechanisms of these CRISPR enzymes. Here we summarize and compare structures of three types of CRISPR enzymes used in nucleic acid detection captured in their respective functional forms and illustrate the current understanding of their activation mechanism.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , ADN/genética , ARN/genéticaRESUMEN
Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. In this chapter, we focus on the methods required to study the biochemistry of this recently discovered cOA signaling pathway. We cover protein expression and purification, synthesis of cOA and its linear analogues, kinetic analysis of cOA synthesis and cOA-stimulated ribonuclease activity, and small molecule detection and identification with thin-layer chromatography and mass spectrometry. The methods described are based on our recent studies of the type III CRISPR system in Sulfolobus solfataricus, but are widely applicable to other type III systems.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleótidos/metabolismo , Sulfolobus solfataricus/metabolismo , Nucleótidos de Adenina/genética , Proteínas Arqueales/genética , Proteínas Asociadas a CRISPR/genética , Clonación Molecular/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Escherichia coli/genética , Cinética , Oligorribonucleótidos/genética , Sistemas de Mensajero Secundario , Transducción de Señal , Sulfolobus solfataricus/genéticaRESUMEN
A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk .
Asunto(s)
Archaea/genética , Bacterias/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Familia de Multigenes , Proteínas Asociadas a CRISPR/química , Mapeo Cromosómico , Eliminación de Gen , Filogenia , Dominios Proteicos , Synechocystis/genéticaRESUMEN
The CRISPR/Cas adaptive immune system shows extreme diversity in the number of CRISPR/Cas types and subtypes, and in the multitude of CRISPR associated protein families of which they are composed. Despite this diversity, the roles of many Cas protein families are now defined with regard to spacer acquisition, crRNA biogenesis, and DNA or RNA surveillance and targeting. However, a number of unclassified CRISPR-Cas proteins remain. Such proteins have traditionally been designated as CRISPR subtype x (Csx). Here we revisit the structural analysis of one such protein, Csx3, and show that this homodimeric protein utilizes a Rossmann fold for the recognition of an RNA tetranucleotide. Tertiary and quaternary structural similarities of Csx3 to CRISPR/Cas proteins Csx1 and Csa3 are identified and suggest Csx3 is a new member of the CRISPR Associated Rossmann Fold (CARF) superfamily. The structure of the Csx3/RNA complex illustrates one way CARF domain proteins may recognize pseudo-symmetric polynucleotides.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Exorribonucleasas/química , Manganeso/química , Conformación Molecular , Ribonucleasas/químicaRESUMEN
Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.