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Prokaryotic Evolution of CRISPR Targeting

Final Report Summary - PROTECT (Prokaryotic Evolution of CRISPR Targeting)

A summary of progress towards objectives and details for each task;
1. To obtain insight into when different resistance mechanisms will be of the greatest benefit, I have competed CRISPR-Cas knockout strains and wild type P. aeruginosa PA14 strains under varying environmental quality (high and low nutrient environments). I have competed the mutant against the wild type in the absence or presence of phages DMS3 that can infect PA14. Each competition has been replicated 6 times, Relative fitness of the strains has been calculated from changes in frequencies. I have established the reasons for phenotypic fitness differences in terms of resistance to phages and costs associated with resistance using standard plaque assays and competition experiments against the wildtype in the presence and absence of phage. These assays together have revealed that CRISPR-Cas adaptive immunity is favoured at low phage exposure, and surface modification-mediated immunity at high phage exposure. I found that this is because surface modification provides constitutive defence and is associated with a constitutive cost of resistance. CRISPR-Cas mediated resistance on the other hand is an inducible defence and is associated with an inducible cost of resistance.

2. I have carried out the proposed coevolution experiments for approximately 700 bacterial generations (100 experimental transfers). Surprisingly, we found that co-evolution is short lived, and phage goes extinct rapidly. This is the opposite of how CRISPR based coevolution is generally thought to proceed, but these insights are based on models and highly simplified bacterial communities. We integrated theoretical analysis with experimental evolution to understand why phage cannot evolve to overcome CRISPR based immunity and fount that the combination of specificity of the infection process coupled to high levels of CRISPR allele diversity is crucial for this effect. These findings are described in a manuscript that has been submitted to Nature.

3. I have monitored spacer acquisition during laboratory evolution experiments using PCR amplification using a primer upstream of the CRISPR and a CRISPR-internal primer. Clones that contained newly acquired spacers, as shown from PCR analysis, have been sequenced. The acquired spacer sequences have been compared with phage sequences and analysed for PAMs adjacent to the target sequence, for strand specificity of spacer acquisition. Sequencing of CRISPR loci also revealed spacer duplications and occasional loss of sequences during the coevolution experiment, which has provided further insight into the dynamics of spacer incorporation and loss during phage challenge. These data are described in (Westra et al, Current Biology 2015).

4. Resequencing of phage genomes using next-generation sequencing to monitor the genetic basis of the phage response to bacterial CRISPR-based resistance is no longer relevant, since phage does not co-evolve for extended periods of time.

A summary of the progress of the researcher training activities/transfer of knowledge activities/integration activities (as it applies for the MC action)

The topic that is dealt with in this research is related to my Ph.D. but approaches it from a different perspective. While my Ph.D. focused on the biochemical characterization of a bacterial immune system, the current research aims at unraveling the functional importance of bacterial immune systems and identifying the conditions where these systems matter. To develop my expertise in the field of coevolution of bacterial hosts and their phages, the scientific training program will contain conferences:

_ EMBO | EMBL Symposium: Evolution and Ecology of Model Organisms – May 2013, Heidelberg, Germany
_ The Evolution Annual Meeting – June 2013, Snowbird, UT, USA
_ European Society for Evolutionary Biology conference, August 2013, Lisbon, Portugal
These are yearly conferences, and can be attended in 2013 or the year after.

_ April 2013 in Exeter: Yearly MMEMS Workshop (Mathematical Modelling and Experimental Microbial Systems), on the impact of microevolution on community structures.
_ NERC next generation bioinformatics courses (introduction to Bio-Linux). 2013 BBSRC (UK) Summer School in Molecular Evolution and Diversity (location to be announced)

Furthermore, the applicant will spend 2 weeks at the University of Liverpool to receive training in next generation sequencing from the staff of the bioinformatics group at the Centre for Genome Research, under the supervision of Professor Steve Paterson.


I have attended the ESEB meeting in 2013 as proposed.
I have attended the Roscoff evolution meeting instead of the EMBO EMBL meeting. The roscoff meeting allowed me to share my exciting new data with a host-pathogen co-evolution audience, and allowed me to interact and network with renowned experts from the field.
I have attended the CRISPR meeting in St Andrews in 2014.
I have attended the CRISPR meeting in New York in 2015.

I have received intensive training in writing grant proposals and panel interviews. This training was given in the context of writing a NERC Independent Research Fellowship proposal, and the interview training followed when I was invited for interview for this proposal. The NERC IRF was successful, leading to a permanent appointment at the University of Exeter as a Lecturer.

Resequencing of phage genomes using next-generation sequencing to monitor the genetic basis of the phage response to bacterial CRISPR-based resistance is no longer relevant, since phage does not co-evolve for extended periods of time. I therefore did not visit the lab of Prof. Patterson.

Highlight clearly significant results;
We obtained the following clearly significant results, from aim1 and aim2 of the proposed research.

In our manuscript (Westra et al, submitted) we use bacteria and their phages to experimentally answer a long standing question in evolutionary biology: what factor(s) have been driving the evolution of the 2 distinct immune strategies that are ubiquitous in nature? (1) inducible defences (e.g. the human adaptive immune system; plant volatiles; insect antimicrobial peptides) and (2) constitutive defences (e.g. the human innate immune system; plant toxins; insect melanisation). By analysing the mechanistic basis of acquired immunity in bacteria in response to different viral exposures we show the evolution of these 2 distinct immune strategies in real-time. Furthermore, our experiments tease apart the underlying mechanism, which allows us to directly attribute the differential evolution of constitutive and inducible defences to differences in parasite exposure, rather than correlating it with environmental factors (e.g. resource availability). In a commentary by Dr. Andrea Graham (Princeton, USA) this work was characterized as “a tour de force empirical test of theory.”

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. In our manuscript submitted to nature, 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 high specificity of infection and greatly increases overall population resistance. Apart from this group benefit, diversity also increases the likelihood that bacterial populations contain highly resistant individual clones. However, the magnitude of the group benefit is as great as the individual benefit, suggesting that multi-level selection has driven the propensity of CRISPR-Cas to generate spacer diversity. We propose that the short-lived nature of CRISPR-dependent bacteria-virus coevolution that results from the inability to overcome CRISPR immunity by point mutation has favored the evolution of sophisticated virus-encoded mechanisms to overcome CRISPR immunity, such as anti-CRISPR proteins that block the CRISPR-Cas machinery.

If applicable, explain the reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning;

If applicable, explain the reasons for failing to achieve critical objectives and/or not being on schedule and explain the impact on other tasks as well as on available resources and planning (the explanations should be coherent with the declaration by the scientist in charge) ;

A statement on the use of resources, in particular highlighting and explaining deviations between actual and planned researcher-months in Annex 1 (Description of Work)
There are no deviations; the project runs according to plan.

If applicable, propose corrective actions.