CRISPR ecology and evolution

09:00-09:05 Welcome by the Royal Society & lead organiser

09:05-09:30 A glimpse of CRISPR

Dr Francisco J M Mojica, University of Alicante, Spain

Abstract

Regularly interspaced DNA repeats had been independently reported in evolutionarily distant prokaryotes before similar structures were related with each other in 2000, when the SRSR type of repeats (subsequently renamed as CRISPR) was defined. Transcription of the CRISPR arrays and initial studies on CRISPR activity were already documented in the early 1990s and CRISPR-associated (Cas) proteins were identified a decade later. However, the role played by CRISPR and Cas was a mystery until the revelation in 2005 that repeat-intervening regions (known as spacers) were retentions of infectious nucleic acids. The apparent incompatibility between the presence of spacers in a genome and perfectly matching sequences (ie, protospacers) elsewhere within the cell, hinted at an involvement of CRISPR in protection against invaders carrying protospacers. Such a surprising discovery, of an adaptive immunity device operating in bacteria and archaea, expedited research on CRISPR-Cas to uncover the basics of this genetic barrier: single-spacer CRISPR RNAs (crRNAs) guide a Cas endonuclease to sequences matching the carried spacer, resulting in target cleavage. A plethora of CRISPR applications, both in the native host and heterologous cells, has emerged from the impressive progress of knowledge made on the biochemistry of these systems during the last decade. Still, general aspects regarding CRISPR biology, in nature, remain to be elucidated thirty years after the existence of these sequences was announced.

09:30-09:45 Discussion

09:45-10:15 Origins of the key components of the CRISPR-Cas systems

Dr Eugene Koonin, National Center for Biotechnology Information (NCBI), USA

Abstract

The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, that employ a homolog of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, Class 1 with multisubunit effectors, and Class 2 in which the effector consists of a single, large protein. The Class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the Class 1 effector complexes remains murky. However, the recent discovery of a signaling pathway built into the type III systems of Class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal-transduction system involved in stress-induced programmed cell death. The subsequent evolution of the Class 1 effector complexes through serial gene duplication and displacement, primarily, of genes for proteins containing RNA Recognition Motif (RRM) domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, Dr Koonin attempts a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution.

10:15-10:30 Discussion

10:30-11:00 Coffee

11:00-11:30 A matter of background: DNA repair pathways as a cause for the sparse distribution of CRISPR-Cas systems in bacteria

Dr Eduardo Rocha, Pasteur Institute, France

Abstract

The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because of their role in adaptive immunity, their frequent transfer between species and their much higher frequency in archaea. Furthermore, restriction-modification systems, often regarded as the innate immunity counterpart of CRISPR-Cas systems, are almost ubiquitous in bacteria. Here, Dr Rocha’s group investigates the possibility that the success of CRISPR-Cas acquisition by horizontal gene transfer is affected by the interactions of these systems with the host genetic background and especially with components of double-strand break repair systems (DSB-RS). Dr Rocha’s group shows that such systems are more often positively or negatively correlated with the frequency of CRISPR-Cas systems than random genes of similar frequency. The detailed analysis of these co-occurrence patterns shows that Dr Rocha’s group method identifies previously known cases of mechanistic interactions between these systems. It also reveals other positive and negative patterns of co-occurrence. Dr Rocha’s group have experimentally tested the negative association between type II-A systems and NHEJ and found them to be caused by lower efficiency of repair by the latter in presence of the former. The researchers also find that the patterns of distribution of CRISPR-Cas systems in Proteobacteria are strongly dependent on the epistatic groups including RecBCD and AddAB. Their results suggest that the genetic background plays an important role in the success of establishment of adaptive immunity in different bacterial clades and provide insights to guide further experimental research on the interactions between CRISPR-Cas and DSB-RS.

11:30-11:45 Discussion

11:45-12:15 Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race

Dr Kimberley Seed, UC Berkeley, USA

Abstract

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defense against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, the researchers exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but the researchers find that single spacers do not equally interfere with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V cholera chromosome. The group finds that targeting the V cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. Dr Seed proposes a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the chromosome before PLE mobilisation. Generally, laboratory acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.

12:15-12:30 Discussion

13:30-14:00 Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers

Associate Professor Peter Fineran, University of Otago, New Zealand

Abstract

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape CRISPR-Cas defence. Spacer acquisition occurs by either naïve or primed adaptation. Naïve adaptation typically results in the incorporation of a single spacer. In contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. The researchers predicted that single and multiple spacers, representative of naïve and primed adaptation respectively, would cause differing outcomes after phage infection. The researchers investigated the response of two phages, ɸTE and ɸM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ɸTE structure revealed shortened tails in escape mutants with tape measure protein deletions. The researchers conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naïve and primed acquisition events.

14:00-14:15 Discussion

14:15-14:45 How microbes keep their CRISPR memories functional and up to date

Dr Stan Brouns, Delft University of Technology, the Netherlands

Abstract

The CRISPR immune system protects bacteria and archaea from invading viruses and plasmids. Immunity depends on protein diverse protein complexes that use small RNA molecules to find matching viral or plasmid DNA. Dr Brouns will show how viruses escape immunity by mutating target sites or glycosylating their DNA, and will highlight a mechanism called priming that updates the CRISPR memory leading to rapid co-evolution between host and phage. Dr Brouns will present a new mechanism catalyzed by Cas4 proteins that selects functional CRISPR memories with a PAM (protospacer adjacent motif) that is essential for efficient CRISPR targeting. We introduced the CRISPR adaptation genes cas1, cas2 and cas4 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host. New spacers displayed variation in spacer length, which is typical for spacers found in the native I-D host and for cas4-containing CRISPR systems in general. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM.

14:45-15:00 Discussion

15:00-15:30 Tea

15:30-16:00 Changes in CRISPR arrays content over time and space

Dr Konstantin Severinov, Waksman Institute, USA, Skolkovo Institute of Science and Technology, Russia

16:00-16:15 Discussion

16:15-16:45 Evolutionary structure of CRISPR-Cas diversity in natural populations

Dr Rachel Whitaker, University of Illinois, USA

Abstract

Dr Whitaker will describe quantitative metrics for measuring CRISPR-Cas diversity and what they can reveal about past and future of immune interactions between viruses and microbes in wild microbial populations. It is Whitaker’s group goal to provide a framework that would allow prediction of virus epidemics in microbial populations. They also aim to understand key parameters that define the stability, diversity and evolutionary trajectory of engineered, natural and clinical microbial populations important to global health and disease.

16:45-17:00 Discussion

17:00-18:00 Poster session

09:00-09:30 Phages and CRISPR-Cas systems: the ongoing battle

Professor Sylvain Moineau, Université Laval, Canada

Abstract

Fighting viruses is no easy task. Bacterial cells have survived phage attacks by evolving sophisticated defence strategies that enable them to thrive even in virus-rich ecosystems. CRISPR-Cas is one of these mechanisms used by microbes to protect against viral infection. Bacterial CRISPR-Cas type II systems function by first incorporating short DNA ‘spacers’, derived from invading defective phage genomes, in the CRISPR array located in their genome. The bacterial CRISPR array is then transcribed and matured into short RNAs, which, by recruiting Cas9 endonuclease, act as surveillance complexes that recognise and cleave subsequent invading matching DNA sequences. The cleavage occurs near a short motif, called the PAM, adjacent to the sequence targeted by the spacer. Phages have evolved counter-tactics to thwart such mechanisms, leading to a so-called biological arms race. For example, phages can bypass CRISPR immunity through point mutation or deletion of the CRISPR target or PAM in their genome as well as by the production of anti-CRISPR proteins (ACRs). Using the Gram-positive dairy bacterium Streptococcus thermophilus as a model, this presentation will recall the roles played by virulent phages in the understanding of CRISPR-Cas systems as well as in the development of industrially relevant phage-resistant bacterial strains. Professor Moineau will also highlight the recent discovery, characterisation and effectiveness of two families of ACRs from S thermophilus phages. The emergence of ACR-containing phages illustrates the ongoing battle between phages and their hosts and that additional approaches are needed to control phages in industrial settings.

09:30-09:45 Discussion

09:45-10:15 How bacteriophages protect themselves from CRISPR

Dr Joe Bondy-Denomy, UC San Francisco, USA

Abstract

Bacterial CRISPR-Cas systems utilise sequence-specific RNA-guided nucleases to defend against bacteriophage (phage) infection. As a counter-measure, numerous phages produce proteins to block the function of the Class 1 CRISPR-Cas systems, which utilise multi-subunit protein complexes to enact immunity. Dr Bondy-Denomy’s group recently developed and utilised novel bioinformatics strategies to identify Class 2 (eg Cas9 and Cas12) anti-CRISPR proteins. The researchers have identified anti-CRISPR proteins encoded by phages and mobile genetic elements from many organisms, including Pseudomonas aeruginosa, Listeria monocytogenes, Moraxella bovoculi and Streptococcus pyogenes, suggesting widespread and common CRISPR-Cas inactivation. More recently, we have identified a phage with a novel mechanism of CRISPR evasion that does not rely on anti-CRISPR proteins, but instead physically segregates its genome from nucleases. Dr Bondy-Denomy will discuss our progress towards understanding these mechanisms that phages deploy to avoid destruction by CRISPR-Cas and how this work benefits CRISPR-Cas technologies.

10:15-10:30 Discussion

10:30-11:00 Coffee

11:00-11:30 CRISPR-Cas immunity leads to a co-evolutionary arms race between Streptococcus thermophilus and lytic phage

Dr Stineke van Houte, University of Exeter, UK

Abstract

CRISPR-Cas is an adaptive prokaryotic immune system that prevents phage infection. By incorporating phage-derived “spacer” sequences into CRISPR loci on the host genome, future infections from the same phage genotype can be recognised and the phage genome cleaved. However, phage can escape CRISPR degradation by mutating the sequence targeted by the spacer, allowing them to re-infect previously CRISPR-immune hosts, and theoretically leading to co-evolution. Previous studies have shown that phage can persist over long periods in populations of Streptococcus thermophilus that can acquire CRISPR-Cas immunity, but it has remained less clear whether this coexistence was due to co-evolution, and if so, what type of co-evolutionary dynamics were involved. In this talk Dr van Houte will discuss results from highly replicated serial transfer experiments her group performed over 30 days with S thermophilus and a lytic phage. Using a combination of phenotypic and genotypic data, the researchers show that CRISPR-mediated resistance and phage infectivity coevolved over time following an arms race dynamic, and that asymmetry between phage infectivity and host resistance within this system eventually causes phage extinction. This work provides further insight into the way CRISPR-Cas systems shape the population and co-evolutionary dynamics of bacteria-phage interactions.

11:30-11:45 Discussion

11:45-12:15 Phage-bacterium co-evolution in aquaculture

Dr Lotta-Riina Sundberg, University of Jyväskylä, Finland

Abstract

The ecological and evolutionary effects CRISPR-Cas immune system are traditionally studied in controlled laboratory conditions. Aquaculture provides semi-natural conditions for examining the role of CRISPR-Cas in phage/bacterium co-evolution in the environment, as the disease surveillance projects together with interest towards phage therapy have led to collections of phage and bacterial isolates. The researchers sampled a flow-through aquaculture setting during 2007-2014, and characterized the co-evolution of populations of phages and their bacterial hosts Flavobacterium columnare at the phenotypic and genetic level. Bacteria were generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Two CRISPR loci in the bacterial host were identified: a type II-C locus (with Cas9) and a type VI-B locus (with Cas13a). Phage-matching spacers appeared in both loci over time. The spacers mostly targeted the terminal end of the phage genomes, which also varied across time, resulting in arms-race-like changes in the protospacers of the coevolving phage population. On several occasions, the introduction of CRISPR spacers to the bacterial population was followed by the appearance of phage isolates with modifications in the corresponding protospacer regions. The researchers’ results demonstrate that aquaculture can provide essential information on the co-evolutionary dynamics between the phage and its host under environmental conditions.

12:15-12:30 Discussion

13:30-14:00 Evolutionary emergence of infectious diseases in heterogeneous host populations

Dr Sylvain Gandon, CNRS Montpellier, France

Abstract

The emergence of pathogens remains a major public health concern. Unfortunately, when and where pathogens will (re-)emerge is notoriously difficult to predict as the erratic nature of those events is reinforced by the stochastic nature of pathogen evolution during the early phase of an epidemic. For instance, mutations allowing pathogens to escape host resistance may boost pathogen spread and promote emergence. Yet the ecological factors that govern such evolutionary emergence remain elusive both because of the lack of ecological realism of current theoretical frameworks and the difficulty of experimentally testing their predictions. The researchers developed a theoretical model to explore the effects of the heterogeneity of the host population on the probability of pathogen emergence, with or without pathogen evolution. The researchers show that evolutionary emergence and the spread of escape mutations in the pathogen population is more likely to occur when the host population contains an intermediate proportion of resistant hosts. This probability of evolutionary emergences rapidly declines with the diversity of resistance in the host population but also with the multiplicity of host resistance per individual host. Experimental tests using lytic bacteriophages infecting their bacterial hosts containing CRISPR-Cas immune defenses confirm these theoretical predictions. Dr Gandon will discuss how these results can help design more effective control strategies against the emergence of infectious diseases.

14:00-14:15 Discussion

14:15-14:45 From the environment to the lab - eco-evolutionary roles of haloarchaeal CRISPR-Cas systems

Dr Uri Gophna, Molecular Cell Biology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Israel

Abstract

Many archaea possess spacers that match chromosomal genes of related species, including those encoding core housekeeping genes. By sequencing genomes of environmental archaea isolated from a single site, Dr Gophna’s group demonstrated that inter-species spacers are common. They then showed experimentally, by mating Haloferax volcanii and Haloferax mediterranei, that spacers are indeed acquired chromosome-wide, although a preference for integrated mobile elements and nearby regions of the chromosome exists. Engineering the chromosome of one species to be targeted by the other’s CRISPR–Cas reduces gene exchange between them substantially. Thus, spacers acquired during inter-species mating could limit future gene transfer, resulting in a role for CRISPR–Cas systems in microbial speciation. More recently the researchers have identified a virus that chronically infects one of their natural Haloferax isolates and can also integrate into its genome. Exposure to this virus elicited strong and specific spacer acquisition by the H volcanii lab strain, that surprisingly could not be stably infected by that virus. This raised the question why the virus' original host that appears to have a functional CRISPR-Cas system did not acquire spacers from a virus that chronically infects it. The researchers addressed this by an “active immunisation” approach in which provirus-containing cells were exposed to mature virus particles. K Höveler, J Deiglmayr and M Beyer from Physical Chemistry Laboratory, ETH Zurich, Switzerland also contributed to the research outlined in this talk.

14:45-15:00 Discussion

15:00-15:30 Tea

15:30-16:00 Possibilities for pest and vector control with CRISPR-based constructs

Professor Austin Burt, Imperial College London, UK

Abstract

In this talk Professor Burt will describe how CRISPR technology may be used to develop new tools for controlling disease vectors and other pest species. The primary focus will be on a particular case study, the development of gene drive approaches to control mosquitoes that transmit malaria in sub-Saharan Africa. Gene drive is a naturally occurring process by which genes are able to bias their transmission from one generation to the next, and can allow a gene to spread through a population even if it is harmful to the organisms carrying it. Synthetic gene drive systems have been developed in mosquitoes using CRISPR technology and proof-of-principle for population control demonstrated in the lab. Issues arising in the further development of this technology will be discussed, and then prospects for CRISPR-based tools to control other pest species.

16:00-16:15 Discussion

16:15-16:45 Understanding the academic debate on genome editing in animals

Dr Sarah Hartley, Exeter University, UK
Nienke de Graeff, University Medical Center Utrecht, the Netherlands

Abstract

In recent years, new genome editing technologies have emerged that can edit the genome of non-human animals with progressively increasing efficiency. Despite ongoing academic debate about the ethical implications of these technologies, no comprehensive overview of this debate exists. To address this gap in the literature, the researchers conducted a systematic review of the reasons reported in the academic literature for and against the development and use of genome editing technologies in animals. Most included articles were written by academics from the biomedical or animal sciences. The reported reasons related to seven themes: human health, efficiency, risks and uncertainty, animal welfare, animal dignity, environmental considerations and public acceptability. The findings illuminate several important considerations about the academic debate, including a relatively low disciplinary diversity in the contributing academics, a scarcity of systematic comparisons of potential consequences of using these technologies, an underrepresentation of animal interests, and a disjunction between the public and academic debate on this topic. As such, this article can be considered a call for a broad range of academics to get increasingly involved in the discussion about genome editing, to incorporate animal interests and systematic comparisons, and to further discuss the aims and methods of public involvement.

16:45-17:00 Discussion