CRISPR-Cas Adaptive Immunity Research Articles

TnpB homologues exapted from transposons are RNA-guided transcription factors.

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Casadaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

The impact of phage and phage resistance on microbial community dynamics.

Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. While our previous work showed how a microbial community may impact phage resistance evolution, little is known about the inverse, namely how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild-type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Additionally, we used mathematical modelling to explore the ecological interactions underlying full community behaviour, as well as to identify general principles governing the impacts of phage on community dynamics. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Finally, we show that pairwise data alone is insufficient when modelling our microbial community, both with and without phage, highlighting the importance of higher order interactions in governing multispecies dynamics in complex communities. Combined, our data clearly illustrate how phage targeting a dominant species allows for the competitive release of the strongest competitor while also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.

Cryo-EM structure of the transposon-associated TnpB enzyme

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR–Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA–target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR–Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR–Cas12 effectors.

High-MOI induces rapid CRISPR spacer acquisition in Sulfolobus from an acr deficient virus.

Spacer acquisition, the first step in CRISPR-Cas adaptive immunity, plays a critical role in establishing and strengthening host defense against mobile genetic elements (MGEs). Here we present a host-virus system, where an increase in the multiplicity of infection (MOI), of a CRISPR-Cas susceptible virus, forces rapid spacer acquisition in the Sulfolobus islandicus LAL14/1 CRISPR arrays. Spacer acquisition was observed as early as 30 minutes post infection, with the newly acquired spacers uniformly distributed across the genome of the virus. Although the newly acquired spacers were predominantly effective only against the CRISPR-Cas susceptible mutant virus, we were able to isolate a host mutant with a novel spacer which provides immunity against the multiple Acr encoding wildtype virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2).

Introduction: the secret lives of microbial mobile genetic elements.

Introduction: the secret lives of microbial mobile genetic elements.

Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli

CRISPR-Cas systems provide immunity against phages and plasmids in bacteria and archaea. Despite the popularity of CRISPR-Cas9 based genome editing, few endogenous systems have been characterized to date. Here, we sampled 1,262 publically available lactobacilli genomes found them to be enriched with CRISPR-Cas adaptive immunity. While CRISPR-Cas is ubiquitous in some Lactobacillus species, CRISPR-Cas content varies at the strain level in most Lactobacillus species. We identified that Type II is the most abundant type across the genus, with II-A being the most dominant sub-type. We found that many Type II-A systems are actively transcribed, and encode spacers that efficiently provide resistance against plasmid uptake. Analysis of various CRISPR transcripts revealed that guide sequences are highly diverse in terms of crRNA and tracrRNA length and structure. Interference assays revealed highly diverse target PAM sequences. Lastly, we show that these systems can be readily repurposed for self-targeting by expressing an engineered single guide RNA. Our results reveal that Type II-A systems in lactobacilli are naturally active in their native host in terms of expression and efficiently targeting invasive and genomic DNA. Together, these systems increase the possible Cas9 targeting space and provide multiplexing potential in native hosts and heterologous genome editing purpose.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems

SummaryBacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Foreign DNA capture during CRISPR–Cas adaptive immunity

Foreign DNA capture during CRISPR–Cas adaptive immunity

Foreign DNA capture during CRISPR-Cas adaptive immunity.

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit, whereas Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting A-T rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyzes spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity.

Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.

The initial stage of CRISPR–Cas immunity involves the acquisition of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved amongst all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å resolution crystal structure of the Cas1–Cas2 complex. Mutations that perturb Cas1–Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Unlike Cas1, active site mutants of Cas2 can still acquire new spacers indicating a non-enzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1–Cas2 complexes specify sites of CRISPR spacer integration.

CRISPR–Cas adaptive immunity and developments in CRISPR–Cas applications: Reply to comments on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems”

Available online at www.sciencedirect.com ScienceDirect Physics of Life Reviews 11 (2014) 149–151 www.elsevier.com/locate/plrev Reply to comments CRISPR–Cas adaptive immunity and developments in CRISPR–Cas applications Reply to comments on “Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems” Edwin L. Cooper ∗ , Nicola Overstreet Laboratory of Comparative Immunology, Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1763, United States Received 13 December 2013; accepted 17 December 2013 Available online 21 December 2013 Communicated by M. Frank-Kamenetskii We appreciate the commentaries received from Stern [1], Pinti and Cossarizza [2], Plagens and Randau [3], Koonin [4], Wanner et al. [5], and Severinov [6], and their observations and insights, which we will now discuss. As is mentioned in many of the commentaries received, the emergence of knowledge about CRISPR–Cas systems marks an important shift in the mindset of immunology. For a long time, eukaryotic immune systems were viewed through the paradigm of self/not-self. This paradigm was rooted in clonal selection and the dual innate and adaptive model [7–9]. With the advent of the danger hypothesis, it became apparent that the self/not-self pattern was not im- pervious to upheaval [7,10]. Similarly, small RNA systems have shifted the perspective of immunology. When RNA systems were first discovered, they were seen as an exception to the suite of well-known protein-recognition adaptive immune defenses. However, in the words of Pinti and Cossarizza, “it is now clear even to us, human immunologists, that RNA-based defense mechanisms are the rule, and not the exception, and that the modern, adaptive immune system has been built on an ancient basement made by RNA” [2]. Stern similarly remarks that the wealth of RNA systems reveals the “general solutions to the problem of par- asites” [1]. The recognition of not-self motifs, infected cell death, and retained memory of past exposures are all mechanisms of immune defense that are observed across the tree of life [1]. Koonin likens small RNA defense mech- anisms to an idea that evolution found “ ‘too good’ to be abandoned” [4]. We agree that the pervasiveness of small RNA systems in prokaryotes and eukaryotes demonstrates the importance of these systems to life throughout the phy- logenetic tree, and speaks to the power of these types of systems. As Koonin states, “there seems to be underlying logic in their evolution that is both universal and simple” [4]. DOI of original article: http://dx.doi.org/10.1016/j.plrev.2013.11.002. DOIs of comments: http://dx.doi.org/10.1016/j.plrev.2013.11.004, http://dx.doi.org/10.1016/j.plrev.2013.12.001, http://dx.doi.org/10.1016/j.plrev.2013.11.012, http://dx.doi.org/10.1016/j.plrev.2013.12.002, http://dx.doi.org/10.1016/j.plrev.2013.12.003, http://dx.doi.org/10.1016/j.plrev.2013.11.015. * Corresponding author. 1571-0645/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.plrev.2013.12.011

Right of admission reserved, no matter the path

Native CRISPR-Cas adaptive immunity systems prevent plasmid conjugation and virus-mediated gene transfer. Zhang et al. have recently reported in Molecular Cell that natural transformation is also limited by a simple endogenous CRISPR system, which would make an optimal candidate tool for functional applications such as genome editing.