Joseph Bondy-Denomy

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.

Multiple mechanisms for CRISPR–Cas inhibition by anti–CRISPR proteins

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR–Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR–Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR–Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase–nuclease and preventing its recruitment to the DNA-bound CRISPR–Cas complex. In vivo, this anti-CRISPR can convert the CRISPR–Cas system into a transcriptional repressor, providing the first example—to our knowledge—of modulation of CRISPR–Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR–Cas function.

A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

ABSTRACT CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts. The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

To acquire or resist: the complex biological effects of CRISPR–Cas systems

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR associated) systems provide a sophisticated adaptive immune system that offers protection against foreign DNA. These systems are widely distributed in prokaryotes and exert an important influence on bacterial behavior and evolution. However, interpreting the biological effects of a CRISPR-Cas system within a given species can be complicated because the outcome of rejecting foreign DNA does not always provide a fitness advantage, as foreign DNA uptake is sometimes beneficial. To address these issues, here we review data pertaining to the potential in vivo costs and benefits of CRISPR-Cas systems, novel functions for these systems, and how they may be inactivated.

The diversity-generating benefits of a prokaryotic adaptive immune system

Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria–virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.

When a virus is not a parasite: the beneficial effects of prophages on bacterial fitness

Most organisms on the planet have viruses that infect them. Viral infection may lead to cell death, or to a symbiotic relationship where the genomes of both virus and host replicate together. In the symbiotic state, both virus and cell potentially experience increased fitness as a result of the other. The viruses that infect bacteria, called bacteriophages (or phages), well exemplify the symbiotic relationships that can develop between viruses and their host. In this review, we will discuss the many ways that prophages, which are phage genomes integrated into the genomes of their hosts, influence bacterial behavior and virulence.

Quorum sensing controls the Pseudomonas aeruginosa CRISPR-Cas adaptive immune system.

Significance The cell–cell communication process, called quorum sensing, activates all three key aspects of the prokaryotic adaptive immune system (termed CRISPR-Cas): expression, activity, and adaptation in the pathogen Pseudomonas aeruginosa. We show that pro- and antiquorum-sensing compounds activate and repress CRISPR-Cas, respectively, suggesting the exciting possibility of a combination quorum-sensing–inhibition-phage therapy cocktail. In P. aeruginosa, quorum-sensing inhibitors repress virulence, making P. aeruginosa more susceptible to elimination by the human immune system, while simultaneously making P. aeruginosa more prone to killing by phage therapy through inhibition of the CRISPR-Cas defense mechanism. Finally, because we show that quorum sensing activates adaptation by the CRISPR-Cas immune system, a quorum-sensing inhibitor should also reduce acquisition of resistance against the administered phage. CRISPR-Cas are prokaryotic adaptive immune systems that provide protection against bacteriophage (phage) and other parasites. Little is known about how CRISPR-Cas systems are regulated, preventing prediction of phage dynamics in nature and manipulation of phage resistance in clinical settings. Here, we show that the bacterium Pseudomonas aeruginosa PA14 uses the cell–cell communication process, called quorum sensing, to activate cas gene expression, to increase CRISPR-Cas targeting of foreign DNA, and to promote CRISPR adaptation, all at high cell density. This regulatory mechanism ensures maximum CRISPR-Cas function when bacterial populations are at highest risk for phage infection. We demonstrate that CRISPR-Cas activity and acquisition of resistance can be modulated by administration of pro- and antiquorum-sensing compounds. We propose that quorum-sensing inhibitors could be used to suppress the CRISPR-Cas adaptive immune system to enhance medical applications, including phage therapies.

Disabling Cas9 by an anti-CRISPR DNA mimic

Natural inhibitors of Cas9 pretend to be DNA and block target binding, and using them in human cells can reduce off-target events. CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9–single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo–electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

Foreign DNA acquisition by the I-F CRISPR–Cas system requires all components of the interference machinery

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.