Prokaryotic Immune System Research Articles

Investigation of direct repeats, spacers and proteins associated with clustered regularly interspaced short palindromic repeat (CRISPR) system of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a ubiquitous bacterium of the marine environment is an important food-borne pathogen responsible for gastroenteritis worldwide. In this study, we aimed to investigate the occurrence and diversity of the CRISPR-Cas system in V. parahaemolyticus genomes using a bioinformatics approach. The CRISPR-Cas system functions as an adaptive immune system in prokaryotes that provides immunity against foreign genetic elements. In total, 570 genomes V. parahaemolyticus genomes were analyzed of which 200 confirmed for the presence of CRISPR-Cas system. The CRISPR-Cas loci were further analyzed for their repeats, spacers and associated Cas proteins. Among the 200 V. parahaemolyticus strains analyzed, 16 (8%) strains possessed the CRISPR-Cas system of complete subtype I-F, while the remaining 184 (92%) harbored the minimalistic type, a subtype I-F variant. Orphan CRISPR repeats and Cas genes were found in one strain each. The CRISPR-associated direct repeat had an unit length of 28 bases. The number of repeat units in each array ranged from 3 to 5 or 5-41 depending on whether they belonged to the minimalistic or complete subtype-IF CRISPR-Cas system, respectively. Of the 768 spacers analyzed in this study, 295 were found to be unique to V. parahaemolyticus. Homology analysis of the conserved spacers revealed matches to plasmids, phages and gut viruses and self chromosomes. Among the CRISPR-associated proteins, Cas5 and Cas7 proteins were found to be conserved. However, variations were seen in the Cas6 protein, which could be grouped into four different types based on their protein length as well as amino acid composition. We present here the diversity and main features of the CRISPR-Cas system in V. parahaemolyticus, which could provide valuable insights in elucidating the role and mechanism of CRISPR/Cas elements in this pathogen.

Insertion sequences in the CRISPR-Cas system regulate horizontal antimicrobial resistance gene transfer in Shigella strains

Multidrug-resistant (MDR) Shigella strains are an enormous threat to public health. Antimicrobial resistance genes are frequently located on plasmids, phages and integrons, which enter bacterial cells by horizontal gene transfer (HGT). CRISPR-Cas systems are adaptive prokaryotic immune systems in bacteria that confer resistance to foreign genetic material such as phages and other mobile genetic elements. However, this may come at a cost of inhibiting the acquisition of other beneficial genes through HGT. This study investigated how Shigella strains regulate the activity of the CRISPR-Cas system spontaneously when they require an exogenous gene necessary for survival. Insertion sequence (IS) elements were identified in cas genes, such as IS600 in cse2, ISSfl2 in cas6e and IS629 in cse1-cas3. The number of spacers in CRISPR-Cas arrays in strains containing an IS was less than that for strains with no IS. Interestingly, fewer spacers were also found in MDR Shigella isolates. Furthermore, an antimicrobial-resistant strain was constructed by electrotransformation of a resistance plasmid in order to detect changes in the CRISPR-Cas system. It was found that the cse2 gene had a new IS (IS600) in the antimicrobial-resistant strain. Bioinformatics analyses showed that the IS600 insertion hotspot was TGC-GGC in the cse2 gene, and the tertiary structure of the Cse2 protein was different with IS600. IS600 caused a five-order of magnitude decrease in relative expression of the cse2 gene. This study sheds mechanistic light on CRISPR-Cas-mediated HGT of antimicrobial resistance genes in Shigella spp. isolates.

Prevalence and characterization of Staphylococcus aureus and Staphylococcus argenteus in chicken from retail markets in China

Staphylococcus aureus (S. aureus) is one of the most common pathogens causing both human and animal infections. Transmission of S. aureus to humans via contaminated food continues to be a health public concern. In the present study, 104 strains including eight MRSA strains were identified from 507 chicken samples (20.5%) in three cities of China. These strains harbored the highest resistance against penicillin (91.4%), followed by tetracycline (64.4%), erythromycin (53.5%), and kanamycin (32.7%). We used spa typing to classify these isolates into 28 types belonging to two lineages including the predominant type t112 (n = 26) and four newly identified spa types. Among the 104 strains, six carried the CRISPR-Cas system, a prokaryotic immune system which protects against foreign genetic elements. Interestingly, rpoB gene sequencing demonstrated that these six initially designated ST2250 strains were in fact S. argenteus, a novel Staphylococcus species genetically closely related to S. aureus. Three Staphylococcus CRIPSR types containing ten spacers identified in these strains have been reported in CRISPR-positive S. aureus. Additionally, 80% of the spacers showed homology to S. aureus phages demonstrating that these conserved spacers were closely related to the phages in the environment of S. argenteus. We speculated that the identical CRISPR types and spacers in both S. argenteus and S. aureus have resulted via exchange of mobile elements between these two species. Emergence of food-borne ST2250 S. argenteus is a potential threat to human public health.

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPR-based applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

Phage Genetic Engineering Using CRISPR⁻Cas Systems.

Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.

On the Origin and Evolutionary Relationships of the Reverse Transcriptases Associated With Type III CRISPR-Cas Systems.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the “single point of origin” model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the “various origins” model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPR-Cas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the “various origins” hypothesis.

Bacterial CRISPR Regions: General Features and their Potential for Epidemiological Molecular Typing Studies.

Introduction:CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci as novel and applicable regions in prokaryotic genomes have gained great attraction in the post genomics era.Methods:These unique regions are diverse in number and sequence composition in different pathogenic bacteria and thereby can be a suitable candidate for molecular epidemiology and genotyping studies. Results:Furthermore, the arrayed structure of CRISPR loci (several unique repeats spaced with the variable sequence) and associated cas genes act as an active prokaryotic immune system against viral replication and conjugative elements. This property can be used as a tool for RNA editing in bioengineering studies.Conclusion:The aim of this review was to survey some details about the history, nature, and potential applications of CRISPR arrays in both genetic engineering and bacterial genotyping studies.

Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of ‘anti-CRISPR’ proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the ‘gene drive’. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.

Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates.

Disrupting a gene to determine its effect on an organism's phenotype is an indispensable tool in molecular biology. Such techniques are critical for understanding how a gene product contributes to the development and cellular identity of organisms. The explosion of genomic sequencing technologies combined with recent advances in genome‐editing techniques has elevated the possibilities of genetic manipulations in numerous organisms in which these experiments were previously not readily accessible or possible. Introducing the next generation of molecular biologists to these emerging techniques is key in the modern biology classroom. This comprehensive review introduces undergraduates to CRISPR/Cas9 editing and its uses in genetic studies. The goals of this review are to explain how CRISPR functions as a prokaryotic immune system, describe how researchers generate mutations with CRISPR/Cas9, highlight how Cas9 has been adapted for new functions, and discuss ethical considerations of genome editing. Additionally, anticipatory guides and questions for discussion are posed throughout the review to encourage active exploration of these topics in the classroom. Finally, the supplement includes a study guide and practical suggestions to incorporate CRISPR/Cas9 experiments into lab courses at the undergraduate level. © 2018 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 46(2):195–205, 2018.

Crazy About CRISPR: An Interview with Francisco Mojica.

Crazy About CRISPR: An Interview with Francisco Mojica.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10.

Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.

Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.

The CRISPR-Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR-Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4-8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.

High-Throughput Characterization of Cascade type I-E CRISPR Guide Efficacy Reveals Unexpected PAM Diversity and Target Sequence Preferences.

Interactions between Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNAs and CRISPR-associated (Cas) proteins form an RNA-guided adaptive immune system in prokaryotes. The adaptive immune system utilizes segments of the genetic material of invasive foreign elements in the CRISPR locus. The loci are transcribed and processed to produce small CRISPR RNAs (crRNAs), with degradation of invading genetic material directed by a combination of complementarity between RNA and DNA and in some cases recognition of adjacent motifs called PAMs (Protospacer Adjacent Motifs). Here we describe a general, high-throughput procedure to test the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system. These studies were followed with reciprocal experiments in which the consequence of CRISPR activity was survival in the presence of a lytic phage. From the combined analysis of the Cascade system, we found that (i) type I-E Cascade PAM recognition is more expansive than previously reported, with at least 22 distinct PAMs, with many of the noncanonical PAMs having CRISPR-interference abilities similar to the canonical PAMs; (ii) PAM positioning appears precise, with no evidence for tolerance to PAM slippage in interference; and (iii) while increased guanine-cytosine (GC) content in the spacer is associated with higher CRISPR-interference efficiency, high GC content (>62.5%) decreases CRISPR-interference efficiency. Our findings provide a comprehensive functional profile of Cascade type I-E interference requirements and a method to assay spacer efficacy that can be applied to other CRISPR-Cas systems.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.

CRISPR Editing Technology in Biological and Biomedical Investigation.

The CRISPR or clustered regularly interspaced short palindromic repeats system is currently the most advanced approach to genome editing and is notable for providing an unprecedented degree of specificity, effectiveness, and versatility in genetic manipulation. CRISPR evolved as a prokaryotic immune system to provide an acquired immunity and resistance to foreign genetic elements such as bacteriophages. It has recently been developed into a tool for the specific targeting of nucleotide sequences within complex eukaryotic genomes for the purpose of genetic manipulation. The power of CRISPR lies in its simplicity and ease of use, its flexibility to be targeted to any given nucleotide sequence by the choice of an easily synthesized guide RNA, and its ready ability to continue to undergo technical improvements. Applications for CRISPR are numerous including creation of novel transgenic cell animals for research, high-throughput screening of gene function, potential clinical gene therapy, and nongene-editing approaches such as modulating gene activity and fluorescent tagging. In this prospect article, we will describe the salient features of the CRISPR system with an emphasis on important drawbacks and considerations with respect to eliminating off-target events and obtaining efficient CRISPR delivery. We will discuss recent technical developments to the system and we will illustrate some of the most recent applications with an emphasis on approaches to eliminate human viruses including HIV-1, JCV and HSV-1 and prospects for the future. J. Cell. Biochem. 118: 3586-3594, 2017. © 2017 Wiley Periodicals, Inc.

Generation of Genomic Deletions (of Rig-I GENE) in Goat Primary Cell Culture Using CRISPR/CAS9 Method

ABSTRACTCRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway. Cells were screened for inactivation of the Rig-Igene and two positive clones were found out of thirty colonies screened. Thus, cells containing Rig-Igene inactivation could be achieved by CRISPR/Cas9 in goat fibroblast cells.

CRISPR‐Cas

ZusammenfassungCRISPR‐Cas ist ein adaptives und vererbbares Immunsystem, mit dem sich Prokaryoten gegen invasive genetische Elemente wie Viren wehren. Es eignet sich aber auch hervorragend zur Manipulation von Genomen. Die Nuklease Cas9 wird durch eine Leit‐RNA zu einem bestimmten DNA‐Abschnitt geführt und fügt dort einen Doppelstrangbruch ein. Dieser wird durch ein zelleigenes Reparatursystem verknüpft, wobei Fehler entstehen, die das Gen zerstören. In unserem Experiment wird ein Knockout des stabil in eine menschliche Glioblastomzelllinie eingefügten, viralen Thymidinsynthase‐Gens durchgeführt. Da letzteres mit dem Gen des Grün Fluoreszierenden Proteins fusioniert ist, lässt sich der Erfolg des Experiments an der abnehmenden grünen Fluoreszenz verfolgen. Gleichzeitig fluoreszieren Zellen, die Cas9 produzieren, orange‐rot, da Cas9 immer zusammen mit dem Orange Fluoreszierenden Protein produziert wird. Aus grün wird rot

CRISPR/CAS9 Technologies.

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) pathway is revolutionizing biological research. Modifications to this primitive prokaryotic immune system now enable scientists to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms. Thus, many laboratories can now perform important experiments that previously were considered scientifically risky or too costly. Here, we describe the components of the CRISPR/Cas system that have been engineered for use in eukaryotes. We also explain how this system can be used to genetically modify cell lines and model organisms, or regulate gene expression in order to search for new participants in biological pathways. © 2017 American Society for Bone and Mineral Research.

HMMCAS: A Web Tool for the Identification and Domain Annotations of CAS Proteins.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immune systems are discovered in many bacteria and most archaea. These systems are encoded by cas (CRISPR-associated) operons that have an extremely diverse architecture. The most crucial step in the depiction of cas operons composition is the identification of cas genes or Cas proteins. With the continuous increase of the newly sequenced archaeal and bacterial genomes, the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here, we describe HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences. HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast, interactive service based on a comprehensive collection of hidden Markov models of Cas protein family. It can accurately identify the Cas proteins including those fusion proteins, for example the Cas1-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B (Cab. thermophilum B). HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.