CRISPR adaptive immunity systems of procaryotes

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a newly identified prokaryotic immunity system against foreign genetic elements. In contrast to other cellular defense mechanisms (e.g. restriction-modification) CRISPR-mediated immunity is adaptive and can be programmed to protect cells against a particular bacteriophage or conjugative plasmid. In this review we describe general principles of CRISPR systems action and summarize known details of CRISPR systems from different microorganisms.     

Target Motifs Affecting Natural Immunity by a Constitutive CRISPR-Cas System in Escherichia coli

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (cas) genes conform the CRISPR-Cas systems of various bacteria and archaea and produce degradation of invading nucleic acids containing sequences (protospacers) that are complementary to repeat intervening spacers. It has been demonstrated that the base sequence identity of a protospacer with the cognate spacer and the presence of a protospacer adjacent motif (PAM) influence CRISPR-mediated interference efficiency. By using an original transformation assay with plasmids targeted by a resident spacer here we show that natural CRISPR-mediated immunity against invading DNA occurs in wild type Escherichia coli. Unexpectedly, the strongest activity is observed with protospacer adjoining nucleotides (interference motifs) that differ from the PAM both in sequence and location. Hence, our results document for the first time native CRISPR activity in E. coli and demonstrate that positions next to the PAM in invading DNA influence their recognition and degradation by these prokaryotic immune systems.     

Advances in detecting and reducing off-target effects generated by CRISPR-mediated genome editing

CRISPR-mediated genome editing is a revolutionary technology for genome manipulation that uses the CRISPR-Cas systems and base editors.Currently,poor efficiency and off-target problems have impeded the application of CRISPR systems.The on-target efficiency has been improved in several advanced versions of CRISPR systems,whereas the off-target detection still remains a key challenge.Here,we outline the different versions of CRISPR systems and off-target detection strategies,discuss the merits and limitations of off-target detection methods,and provide potential implications for further gene editing research.     

Identifying your enemies – could envelope stress trigger microbial immunity?

Microbes utilize defence systems with fundamental similarities to our innate and adaptive immune responses to protect themselves from harmful invaders. One system, made up of CRISPR loci & Cas proteins, incorporates recognizable features from the genomes of viruses (bacteriophages) and plasmids into bacterial genomes, where they are later used to direct a ribonucleoprotein complex to destroy invading nucleic acids upon re-exposure. CRISPR-mediated defence against invasive nucleic acids is found in most archaea and many eubacteria. Many aspects of this newly described defence system have not been worked out, including the molecular mechanisms by which foreign nucleic acids are incorporated into microbial genomes during adaption and destroyed during interference. In this issue of Molecular Microbiology, DeLisa and colleagues provide insight into how this form of microbial immunity might be regulated in eubacteria. They demonstrate that Escherichia coli CRISPR-mediated immunity requires the presence of the BaeSR two-component system under certain conditions. Since BaeSR regulate an envelope stress response, their data imply that immunity against invading, foreign nucleic acids may be somehow linked to stresses to the bacterial membrane. These observations will help pave the way to understanding how and when CRISPR-based immunity may be important in driving evolution and adaptation in eubacteria.     

Nasty viruses, costly plasmids, population dynamics, and the conditions for establishing and maintaining CRISPR-mediated adaptive immunity in bacteria

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.     

Regulated CRISPR Modules Exploit a Dual Defense Strategy of Restriction and Abortive Infection in a Model of Prokaryote-Phage Coevolution

CRISPRs offer adaptive immunity in prokaryotes by acquiring genomic fragments from infecting phage and subsequently exploiting them for phage restriction via an RNAi-like mechanism. Here, we develop and analyze a dynamical model of CRISPR-mediated prokaryote-phage coevolution that incorporates classical CRISPR kinetics along with the recently discovered infection-induced activation and autoimmunity side effects. Our analyses reveal two striking characteristics of the CRISPR defense strategy: that both restriction and abortive infections operate during coevolution with phages, driving phages to much lower densities than possible with restriction alone, and that CRISPR maintenance is determined by a key dimensionless combination of parameters, which upper bounds the activation level of CRISPRs in uninfected populations. We contrast these qualitative observations with experimental data on CRISPR kinetics, which offer insight into the spacer deletion mechanism and the observed low CRISPR prevalence in clinical isolates. More generally, we exploit numerical simulations to delineate four regimes of CRISPR dynamics in terms of its host, kinetic, and regulatory parameters.     

CRISPR-mediated host genomic DNA damage is efficiently repaired through microhomology-mediated end joining in Zymomonas mobilis

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against mobile genetic elements(MGEs) through uptake of invader-derived spacers. De novo adaptation samples spacers from both invaders and hosts, whereas primed adaptation shows higher specificity to sample spacers from invaders in many model systems as well as in the subtype I-F system of Zymomonas mobilis. Self-derived spacers will lead to CRISPR self-interference. However, our in vivo study demonstrated that this species used the microhomology-mediated end joining(MMEJ) pathway to efficiently repair subtype I-F CRISPR-Cas system-mediated DNA breaks guided by the self-targeting spacers. MMEJ repair of DNA breaks requires direct microhomologous sequences flanking the protospacers and leads to DNA deletions covering the protospacers. Importantly, CRISPR-mediated genomic DNA breaks failed to be repaired via MMEJ pathway in presence of higher copies of short homologous DNA. Moreover, CRISPR-cleaved exogenous plasmid DNA was failed to be repaired through MMEJ pathway, probably due to the inhibition of MMEJ by the presence of higher copies of the plasmid DNA in Z. mobilis. Our results infer that MMEJ pathway discriminates DNA damages between in the host chromosome versus mobile genetic element(MGE) DNA, and maintains genome stability post CRISPR immunity in Z. mobilis.     

Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.Subject terms: Bacteriophages, Microbial ecology     

Persisting viral sequences shape microbial CRISPR-based immunity

Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This 'trailer-end conservation' occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. 'Trailer-end clonality' occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining 'trailer-end conservation,' we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.     

CRISPR-mediated phage resistance and the ghost of coevolution past

The past is never dead. It''s not even pastWilliam Faulkner (1951)

Bacteria can acquire heritable immunity to viral (phage) enemies by incorporating phage DNA into their own genome. This mechanism of anti-viral defence, known by the acronym CRISPR, simultaneously stores detailed information about current and past enemies and the evolved resistance to them. As a high-resolution genetic marker that is intimately tied with the host–pathogen interaction, the CRISPR system offers a unique, and relatively untapped, opportunity to study epidemiological and coevolutionary dynamics in microbial communities that were previously neglected because they could not be cultured in the laboratory. We briefly review the molecular mechanisms of CRISPR-mediated host–pathogen resistance, before assessing their potential importance for coevolution in nature, and their utility as a means of studying coevolutionary dynamics through metagenomics and laboratory experimentation.     

Therapeutic applications of CRISPR/Cas9 system in gene therapy

Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. The clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) system, derived from the acquired immune system in bacteria and archaea, has provided a new tool for accurate manipulation of genomic sequence to attain a therapeutic result. The advantage of CRISPR which made it an easy and flexible tool for diverse genome editing purposes is that a single protein (Cas9) complex with 2 short RNA sequences, function as a site-specific endonuclease. Recently, application of CRISPR/Cas9 system has become popular for therapeutic aims such as gene therapy. In this article, we review the fundamental mechanisms of CRISPR-Cas9 function and summarize preclinical CRISPR-mediated gene therapy reports on a wide variety of disorders.     

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.     

CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond

Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.

CRISPR-Cas systems unlock the potential of understanding the molecular basis of plant virus interactions, engineering immunity against plant viruses, and developing sensitive and specific diagnostics.     

Germline Gene Editing in Chickens by Efficient CRISPR-Mediated Homologous Recombination in Primordial Germ Cells

The CRISPR/Cas9 system has been applied in a large number of animal and plant species for genome editing. In chickens, CRISPR has been used to knockout genes in somatic tissues, but no CRISPR-mediated germline modification has yet been reported. Here we use CRISPR to target the chicken immunoglobulin heavy chain locus in primordial germ cells (PGCs) to produce transgenic progeny. Guide RNAs were co-transfected with a donor vector for homology-directed repair of the double-strand break, and clonal populations were selected. All of the resulting drug-resistant clones contained the correct targeting event. The targeted cells gave rise to healthy progeny containing the CRISPR-targeted locus. The results show that gene-edited chickens can be obtained by modifying PGCs in vitro with the CRISPR/Cas9 system, opening up many potential applications for efficient genetic modification in birds.     

In vitro reconstitution of Cascade‐mediated CRISPR immunity in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR)‐encoded immunity in Type I systems relies on the Cascade (CRISPR‐associated complex for antiviral defence) ribonucleoprotein complex, which triggers foreign DNA degradation by an accessory Cas3 protein. To establish the mechanism for adaptive immunity provided by the Streptococcus thermophilus CRISPR4‐Cas (CRISPR‐associated) system (St‐CRISPR4‐Cas), we isolated an effector complex (St‐Cascade) containing 61‐nucleotide CRISPR RNA (crRNA). We show that St‐Cascade, guided by crRNA, binds in vitro to a matching proto‐spacer if a proto‐spacer adjacent motif (PAM) is present. Surprisingly, the PAM sequence determined from binding analysis is promiscuous and limited to a single nucleotide (A or T) immediately upstream (?1 position) of the proto‐spacer. In the presence of a correct PAM, St‐Cascade binding to the target DNA generates an R‐loop that serves as a landing site for the Cas3 ATPase/nuclease. We show that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner. These findings establish a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems.     

Defense Systems Up: Structure of Subtype I-C/Dvulg CRISPR/Cas

The study of CRISPR/Cas systems for RNA-based prokaryotic immunity has emerged as a rapidly expanding frontier in RNA biology. In this issue of Structure, Nam and colleagues provide new clues to deciphering these complex systems in the characterization of a subtype I-C CRISPR/Cas complex.