Review
CRISPR-based adaptive and heritable immunity in prokaryotes

https://doi.org/10.1016/j.tibs.2009.05.002Get rights and content

The recently discovered CRISPR (clustered regularly interspaced short palindromic repeat) defense system protects bacteria and archaea against mobile genetic elements. This immunity system has the potential to continuously adjust its reach at the genomic level, implying that both gain and loss of information is inheritable. The CRISPR system consists of typical stretches of interspaced repetitive DNA (CRISPRs) and associated cas genes. Three distinct stages are recognized in the CRISPR defense mechanism: (i) adaptation of the CRISPR via the integration of short sequences of the invaders as spacers; (ii) expression of CRISPRs and subsequent processing to small guide RNAs; and (iii) interference of target DNA by the crRNA guides. Recent analyses of key Cas proteins indicate that, despite some functional analogies, this fascinating prokaryotic system shares no phylogenetic relation with the eukaryotic RNA interference system.

Section snippets

Mobile genetic elements – opportunities and threads

The analyses of complete genome sequences of bacteria, archaea and eukaryotes have revealed that recombination by horizontal gene transfer has made a major contribution to genome evolution 1, 2. The effect of gaining new genetic material on a host's fitness can be positive, neutral or negative. Hence, a delicate balance has to be established between, on the one hand, DNA uptake and integration systems (e.g. natural competence, conjugation, recombination) and, on the other hand, defense systems

CRISPR–Cas diversity – variations on a theme

Comparative genomics analyses reveal that there is a huge variation in genetic composition, both quantitatively and qualitatively, among CRISPR–Cas systems in different prokaryotes 22, 23 (Figure 2a). An obvious complication in bioinformatics analysis of CRISPR–Cas systems is the fact that there is no guarantee that a particular system is still functional; therefore, some general criteria for functionality have been proposed, including the presence of a leader sequence [24], of invariant repeat

CRISPR mechanism

Three distinct functional stages can be distinguished in the CRISPR–Cas mechanism: (i) CRISPR adaptation, the recognition of alien DNA by dedicated Cas proteins and/or host proteins, as well as the subsequent processing and integration into the chromosomal CRISPR locus; (ii) CRISPR expression, the transcription of the poly-spacer precursor crRNA (pre-crRNA), which is followed by binding to a complex of Cas proteins (Cascade [Cas-complex for anti-virus defense]) and processing to mono-spacer

Remaining questions

The discovery of the CRISPR defense system has generated a lot of excitement. Despite some major breakthroughs that are reviewed here, many questions on the structure and function of its key components remain to be answered. One important matter concerns the recognition of DNA from a genetic element before integration as a CRISPR spacer. There are several possible sites for recognition, including: (i) the PAM in close proximity to the potential proto-spacer 36, 37, 38, 39 (this obviously

Concluding remarks

Despite some similarities with the eukaryotic systems for adaptive immunity (adaptation, memory) and RNA-based control (small guide RNA), the prokaryotic CRISPR–Cas system is substantially different: there is no sequence homology of the key proteins, and DNA rather than RNA is the prime candidate for target interference. Although many details remain to be discovered, it has become clear that the CRISPR–Cas system is a specific, dynamic and inheritable protection system in prokaryotes that has

Acknowledgements

The authors thank Blake Wiedenheft (University of California, Berkeley, USA) for sharing data before publication; Michael and Rebecca Terns (University of Georgia, USA) for Cas6 structure coordinates; and Kira Makarova (National Center for Biotechnology Information, USA), Gerhart Wagner (Uppsala University, Sweden) and Mark Young (Montana State University, USA) for stimulating discussion.

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