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Bacteria and their viruses have coevolved for billions of years. This ancient and still ongoing arms race has led bacteria to develop a vast antiphage arsenal. The development of high-throughput screening methods expanded our knowledge of defence systems from a handful to more than a hundred systems, unveiling many different molecular mechanisms. These findings reveal that bacterial immunity is much more complex than previously thought. In this Review, we explore recently discovered bacterial antiphage defence systems, with a particular focus on their molecular diversity, and discuss the ecological and evolutionary drivers and implications of the existing diversity of antiphage defence mechanisms.In this Review, Georjon and Bernheim provide an overview of the molecular diversity of the most recently discovered bacterial antiphage defence systems and discuss their evolution and the ecological impact of their diversity.

The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR–Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR–Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR–Cas.The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. In this Review, Koonin and colleagues provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on major developments, and outline a complete scenario for the origins and evolution of CRISPR–Cas systems.

Viruses and their hosts are engaged in a constant arms race leading to the evolution of antiviral defence mechanisms. Recent studies have revealed that the immune arsenal of bacteria against bacteriophages is much more diverse than previously envisioned. These discoveries have led to seemingly contradictory observations: on one hand, individual microorganisms often encode multiple distinct defence systems, some of which are acquired by horizontal gene transfer, alluding to their fitness benefit. On the other hand, defence systems are frequently lost from prokaryotic genomes on short evolutionary time scales, suggesting that they impose a fitness cost. In this Perspective article, we present the ‘pan-immune system’ model in which we suggest that, although a single strain cannot carry all possible defence systems owing to their burden on fitness, it can employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains. Thus, the ‘effective’ immune system is not the one encoded by the genome of a single microorganism but rather by its pan-genome, comprising the sum of all immune systems available for a microorganism to horizontally acquire and use.In this Perspective article, Bernheim and Sorek present the ‘pan-immune system’ model in which bacteria employ horizontal gene transfer to access immune defence mechanisms encoded by closely related strains, and conclude by discussing the implications on the evolution of anti-defence strategies in phages.

Key Points CRISPR–Cas systems form two major classes that differ in the organization of their effector modules. The effector modules of class 2 systems consist of a single large protein, which makes them the best candidates for genome-editing tools. Computational methods of microbial genome screening were developed for the comprehensive identification of class 2 CRISPR– cas loci. Using these approaches, six new subtypes of the class 2 system were discovered, which brings the total for this class to three types and 10 subtypes. Type II and type V CRISPR–Cas effectors are homologues of TnpB proteins, which are a poorly characterized family of nucleases that are encoded by bacterial and archaeal transposons. The different subtypes of these two types seem to have evolved independently, through the integration of TnpB-encoding transposons near CRISPR arrays. Type VI effectors are large proteins that contain two RNase domains of the higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) superfamily and that have been shown to, or are predicted to, specifically target RNA. The diverse class 2 CRISPR–Cas systems that have been discovered provide opportunities for the construction of versatile genome-editing tools. Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein. In this Analysis, using a computational pipeline, the authors discover three novel families of class 2 effectors that correspond to three new CRISPR–Cas subtypes and present a comprehensive census of class 2 systems that are encoded in complete and draft bacterial and archaeal genomes. Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR–Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR–Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR–Cas.

CRISPR–Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR–Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR–Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR–Cas systems and mobile genetic elements, including the origins and evolution of CRISPR–Cas systems from DNA transposons, retrotransposons and toxin–antitoxin modules. We discuss how the evolution and structural diversity of CRISPR–Cas systems explain their functional complexity and utility as genome editing tools.CRISPR–Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, Wang, Pausch and Doudna examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR–Cas immune systems and deployed for wide-ranging genome editing applications.

CRISPR–Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR–Cas systems sense and cleave viral DNA, type III and type VI CRISPR–Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR–Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.In this Review, Brouns and colleagues discuss our current understanding of RNA-targeting type III and type VI CRISPR–Cas systems by detailing their composition, properties and defence processes, and describing the biological rationale behind the broad activated immune responses as an effective strategy to combat viral infection.

Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can extract analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extraction and real-time monitoring for macromolecule biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an on-line wearable microneedle patch for extraction and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The experimental results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis. Real-time sensing of biomarkers via the use of wearable devices is a major aim of personalised medicine. Here, authors demonstrate an on-line wearable microneedle patch for real-time capture and monitoring of universal cell-free DNA.

The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called ‘APC-Cas’) that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 10 5 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S . Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens. The detection of pathogens in food and clinical samples remains a challenge. Here, Shen et al. present a detection system, involving a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components, that can detect very low numbers of a bacterial pathogen in milk and serum samples without isolation.

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development. Biocontainment is a key to developing safe genetically-engineered microbes (GEMs). Here the authors demonstrate genetically stable CRISPR-based kill switches that control GEMs’ viability in animal hosts, enabling their safe biomedical applications.

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible. Here, the authors show that sequential treatment with long-acting slow-effective release ART and AAV9- based delivery of CRISPR-Cas9 results in undetectable levels of virus and integrated DNA in a subset of humanized HIV-1 infected mice. This proof-of-concept study suggests that HIV-1 elimination is possible.