Clustered Regularly Interspaced Short Palindromic Repeats Cas Systems Research Articles

Advances in CRISPR therapeutics.

The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR–Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.

CRISPR-CAS9 Sisteminde Kullanılan Polimer Bazlı Transfeksiyon Ajanları

Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) genes and their corresponding CRISPR sequences constitute CRISPR-Cas systems. Due to its simplicity, it is likely that the CRISPR–Cas system could be used effectively in ex vivo gene therapy studies in humans. If this happens, the importance of CRISPR carrier systems will gradually increase. Viral and non-viral systems are used as delivery modalities in genome-editing studies. It has been proven that nanoparticles are the most promising tools for gene therapy due to their adjustable size, surface, shape, and biological behaviours. The polymeric carrier system has become the main non-viral substitute for gene delivery due to its reduced immunogenicity and pathogenicity. In this review, information about current studies related to polymeric carriers used in non-viral CRISPR delivery systems is presented.

Potential Use of CRISPR/Cas13 Machinery in Understanding Virus-Host Interaction.

Prokaryotes have evolutionarily acquired an immune system to fend off invading mobile genetic elements, including viral phages and plasmids. Through recognizing specific sequences of the invading nucleic acid, prokaryotes mediate a subsequent degradation process collectively referred to as the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) (CRISPR–Cas) system. The CRISPR–Cas systems are divided into two main classes depending on the structure of the effector Cas proteins. Class I systems have effector modules consisting of multiple proteins, while class II systems have a single multidomain effector. Additionally, the CRISPR–Cas systems can also be categorized into types depending on the spacer acquisition components and their evolutionary features, namely, types I–VI. Among CRISPR/Cas systems, Cas9 is one of the most common multidomain nucleases that identify, degrade, and modulate DNA. Importantly, variants of Cas proteins have recently been found to target RNA, especially the single-effector Cas13 nucleases. The Cas13 has revolutionized our ability to study and perturb RNAs in endogenous microenvironments. The Cas13 effectors offer an excellent candidate for developing novel research tools in virological and biotechnological fields. Herein, in this review, we aim to provide a comprehensive summary of the recent advances of Cas13s for targeting viral RNA for either RNA-mediated degradation or CRISPR–Cas13-based diagnostics. Additionally, we aim to provide an overview of the proposed applications that could revolutionize our understanding of viral–host interactions using Cas13-mediated approaches.

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR–Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR–Cas-based nucleic acid detection strategies and the application of CRISPR–Cas as a potential antiviral agent in the context of COVID-19.

Investigation into the prevalent CRISPR-Cas systems among the Aeromonas genus.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a prokaryotic adaptive immune system that checks invasion by mobile genetic elements through nuclease targeting. In this study, we investigated the occurrence, diversity, and features of the CRISPR system in the genus Aeromonas using bioinformatics tools. Only 13 out of 122 complete genomes (10.66%) of the genus Aeromonas from the NCBI GenBank database harbored the CRISPR system. The Type I-F system was the most prevalent CRISPR system among the Aeromonads, followed by the Type I-E system. Only one strain harbored a Type I-C CRISPR system. Among the Aeromonads, Aeromonas caviae (22.7%) and Aeromonas veronii (20%) had a higher prevalence rate of the complete CRISPR system. The analysis of direct repeat (DR) sequences showed that all could form stable RNA secondary structures. A phylogenetic tree generated for the Cas1 protein classified CRISPR subtypes into three distinct clusters. Among the 748 spacers investigated, 41.98% and 17.25% showed perfect homology to phage and plasmid sequences, respectively. Some arrays had duplicated spacers. The CRISPR loci are closely linked to antibiotic resistance genes in most strains. Collectively, our results would contribute to research on antibiotic resistance in the Aeromonas group, and provide new insights into the diversity and evolution of the CRISPR-Cas system.

CRISPR-Cas system: a precise tool for plant genome editing

Biotic and abiotic stress affects the productivity and quality of the plant; it is necessarily important to develop improved crops that can withstand the environmental conditions. Conventional methods of breeding are very tedious process that require some alternative. The molecular approach of genetic modification emerged out as a precise method for plant breeding in the past few years. Genome is altered by removing, adding or substituting DNA bases in the host genome. Different methods of genome editing have been developed, including transcription activator like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. These nucleases have enabled researchers to manipulate gene of interest in crop plants. Among these CRISPR-Cas has been developed recently and found to be a more efficient tool for genome editing because of its simple design and low cost. In recent years, CRISPR-Cas9 has been utilized for gene knockouts and knock-ins, multiplex gene targeting, and gene regulation in plants. In this review, we provide the use of CRISPR–Cas9 technology in genome editing and its fruitful applications in plants specifically in terms of improvement in quality and resistance to biotic and abiotic stress. Designing of an efficient CRISPR–Cas system using bioinformatic tools have also been discussed.

The Versatile Type V CRISPR Effectors and Their Application Prospects.

The class II clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR–Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.

CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR–Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR–Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR–Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR–Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR–Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR–Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

Various Aspects of a Gene Editing System-CRISPR-Cas9.

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems

Robust developments of personalized medicine for next-generation healthcare highlight the need for sensitive and accurate point-of-care platforms for quantification of disease biomarkers. Broad presentations of clustered regularly interspaced short palindromic repeats (CRISPR) as an accurate gene editing tool also indicate that the high-specificity and programmability of CRISPR system can be utilized for the development of biosensing systems. Herein, we present a CRISPR Cas system enhanced electrochemical DNA (E-DNA) sensor with unprecedented sensitivity and accuracy. The principle of the E-DNA sensor is the target induced conformational change of the surface signaling probe (containing an electrochemical tag), leading to the variation of the electron transfer rate of the electrochemical tag. With the introduction of CRISPR cleavage activity into the E-DNA sensor, a more apparent signal change between with and without the presence of the target can be achieved. We compared the performance of Cas9 and Cas12a enhanced E-DNA sensor and optimized the chemical environment of CRISPR, achieving a femto-molar detection limit without enzymatic amplification. Moreover, we correlated the CRISPR cleavage signal with the original E-DNA signal as a strategy to indicate potential mismatches in the target sequence. Comparing with classic DNA electrochemistry based mutation detection strategy, CRISPR enhanced E-DNA sensor can determine the presence of a single mutation at an unknown concentration condition. Overall, we believe that the CRISPR enhanced E-DNA sensing strategy will be of especially high utility for point-of-care systems owing to the programmability, modularity, high-sensitivity and high-accuracy.

RNA-Targeting CRISPR–Cas Systems and Their Applications

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR–Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR–Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.

Genome-Wide Analyses Revealed Remarkable Heterogeneity in Pathogenicity Determinants, Antimicrobial Compounds, and CRISPR-Cas Systems of Complex Phytopathogenic Genus Pectobacterium.

The Pectobacterium genus comprises pectolytic enterobacteria defined as the causal agents of soft rot, blackleg, and aerial stem rot diseases of potato and economically important crops. In this study, we undertook extensive genome-wide comparative analyses of twelve species that conform the Pectobacterium genus. Bioinformatics approaches outlined a low nucleotide identity of P. parmentieri and P. wasabiae with other species, while P. carotovorum subsp. odoriferum was shown to harbor numerous pseudogenes, which suggests low coding capacity and genomic degradation. The genome atlases allowed for distinguishing distinct DNA structures and highlighted suspicious high transcription zones. The analyses unveiled a noteworthy heterogeneity in the pathogenicity determinants. Specifically, phytotoxins, polysaccharides, iron uptake systems, and the type secretion systems III–V were observed in just some species. Likewise, a comparison of gene clusters encoding antimicrobial compounds put in evidence for high conservation of carotovoricin, whereas a few species possessed the phenazine, carbapenem, and carocins. Moreover, three clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems: I-E, I-F, and III-A were identified. Surrounding some CRISPR-Cas regions, different toxin and antitoxin systems were found, which suggests bacterial suicide in the case of an immune system failure. Multiple whole-genome alignments shed light on to the presence of a novel cellobiose phosphotransferase system (PTS) exclusive to P. parmenteri, and an unreported T5SS conserved in almost all species. Several regions that were associated with virulence, microbe antagonism, and adaptive immune systems were predicted within genomic islands, which underscored the essential role that horizontal gene transfer has imparted in the dynamic evolution and speciation of Pectobacterium species. Overall, the results decipher the different strategies that each species has developed to infect their hosts, outcompete for food resources, and defend against bacteriophages. Our investigation provides novel genetic insights that will assist in understanding the pathogenic lifestyle of Pectobacterium, a genus that jeopardizes the agriculture sustainability of important crops worldwide.

Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR–Cas systems are classified into different classes and types. Class 1 CRISPR–Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

Molecular mechanisms of III-B CRISPR–Cas systems in archaea

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems provide the adaptive antiviral immunity against invasive genetic elements in archaea and bacteria. These immune systems are divided into at least six different types, among which Type III CRISPR–Cas systems show several distinct antiviral activities as demonstrated from the investigation of bacterial III-A and archaeal III-B systems in the past decade. First, although initial experiments suggested that III-A systems provided DNA interference activity, whereas III-B system was active only in RNA interference, these immune systems were subsequently found to mediate the transcription-dependent DNA interference and the dual DNA/RNA interference. Second, their ribonucleoprotein (RNP) complexes show target RNA (tgRNA) cleavage by a ruler mechanism and RNA-activated indiscriminate single-stranded DNA cleavage, the latter of which is subjected to spatiotemporal regulation such that the DNase activity occurs only at the right place in the right time. Third, RNPs of Type III systems catalyse the synthesis of cyclic oligoadenylates (cOAs) that function as second messengers to activate Csm6 and Csx1, both of which are potent Cas accessory RNases after activation. To date, Type III CRISPR systems are the only known antiviral immunity that utilizes multiple interference mechanisms for antiviral defence.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

CRISPR-Cas systems provide protection against viral1 and plasmid2 infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host1. These sequences, known as spacers, are transcribed into short RNA guides3–5 that specify the cleavage site of Cas nucleases in the genome of the invader6–8. When spacer sequences are acquired during viral infection is not known. To investigate this, we followed spacer acquisition in Staphylococcus aureus cells harboring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers are acquired immediately following infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures the success of the CRISPR immune response.

The new CRISPR-Cas system: RNA-guided genome engineering to efficiently produce any desired genetic alteration in animals.

The CRISPR-Cas system is the newest targeted nuclease for genome engineering. In less than 1year, the ease, robustness and efficiency of this method have facilitated an immense range of genetic modifications in most model organisms. Full and conditional gene knock-outs, knock-ins, large chromosomal deletions and subtle mutations can be obtained using combinations of clustered regularly interspaced short palindromic repeats (CRISPRs) and DNA donors. In addition, with CRISPR-Cas compounds, multiple genetic modifications can be introduced seamlessly in a single step. CRISPR-Cas not only brings genome engineering capacities to species such as rodents and livestock in which the existing toolbox was already large, but has also enabled precise genetic engineering of organisms with difficult-to-edit genomes such as zebrafish, and of technically challenging species such as non-human primates. The CRISPR-Cas system allows generation of targeted mutations in mice, even in laboratories with limited or no access to the complex, time-consuming standard technology using mouse embryonic stem cells. Here we summarize the distinct applications of CRISPR-Cas technology for obtaining a variety of genetic modifications in different model organisms, underlining their advantages and limitations relative to other genome editing nucleases. We will guide the reader through the many publications that have seen the light in the first year of CRISPR-Cas technology.

The basic building blocks and evolution of CRISPR–Cas systems

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) is an adaptive immunity system in bacteria and archaea that functions via a distinct self/non-self recognition mechanism that involves unique spacers homologous with viral or plasmid DNA and integrated into the CRISPR loci. Most of the Cas proteins evolve under relaxed purifying selection and some underwent dramatic structural rearrangements during evolution. In many cases, CRISPR-Cas system components are replaced either by homologous or by analogous proteins or domains in some bacterial and archaeal lineages. However, recent advances in comparative sequence analysis, structural studies and experimental data suggest that, despite this remarkable evolutionary plasticity, all CRISPR-Cas systems employ the same architectural and functional principles, and given the conservation of the principal building blocks, share a common ancestry. We review recent advances in the understanding of the evolution and organization of CRISPR-Cas systems. Among other developments, we describe for the first time a group of archaeal cas1 gene homologues that are not associated with CRISPR-Cas loci and are predicted to be involved in functions other than adaptive immunity.

Requirements for a successful defence reaction by the CRISPR–Cas subtype I-B system

Uptake of foreign mobile genetic elements is often detrimental and can result in cell death. For protection against invasion, prokaryotes have developed several defence mechanisms, which take effect at all stages of infection; an example is the recently discovered CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immune system. This defence system directly degrades invading genetic material and is present in almost all archaea and many bacteria. Current data indicate a large variety of mechanistic molecular approaches. Although almost all archaea carry this defence weapon, only a few archaeal systems have been fully characterized. In the present paper, we summarize the prerequisites for the detection and degradation of invaders in the halophilic archaeon Haloferax volcanii. H. volcanii encodes a subtype I-B CRISPR-Cas system and the defence can be triggered by a plasmid-based invader. Six different target-interference motifs are recognized by the Haloferax defence and a 9-nt non-contiguous seed sequence is essential. The repeat sequence has the potential to fold into a minimal stem-loop structure, which is conserved in haloarchaea and might be recognized by the Cas6 endoribonuclease during the processing of CRISPR loci into mature crRNA (CRISPR RNA). Individual crRNA species were present in very different concentrations according to an RNA-Seq analysis and many were unable to trigger a successful defence reaction. Recognition of the plasmid invader does not depend on its copy number, but instead results indicate a dependency on the type of origin present on the plasmid.

The RNA- and DNA-targeting CRISPR–Cas immune systems of Pyrococcus furiosus

Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5' tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA-Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR-Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR-Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR-Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR-Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.

Hot and crispy: CRISPR–Cas systems in the hyperthermophile Sulfolobus solfataricus

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus.