Welcome by the Royal Society & lead organiser
A glimpse of CRISPR
Dr Francisco J M Mojica, University of Alicante, Spain
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.
Origins of the key components of the CRISPR-Cas systems
Dr Eugene Koonin, National Center for Biotechnology Information (NCBI), USA
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.
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
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.
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
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.