To ensure their survival and persistence in the environment, prokaryotes have developed several defense strategies against invading genetic elements, such as viruses and plasmids. Innate defense mechanisms have been known for years and include restriction modification systems, the alteration of virus receptors on the cell surface, and the secretion of extracellular polymers that prevent virus attachment (1
). Recently, an invader-specific and adaptive defense system was discovered (2
) and named after its typical arrangement of sequence repeats, i.e.
The repeats generally occur nearby a group of protein-coding genes named C
) genes (3
). Recently, CRISPR/Cas systems were classified into three major types and several subtypes based on Cas protein sequences (4
CRISPR/Cas-mediated immunity is achieved via three phases: adaptation, expression, and interference. In the first stage, a piece of the invader DNA is integrated as new spacer into the 5′-end of the CRISPR locus. Transcription of the CRISPR gene in the expression stage produces a long primary CRISPR RNA (pre-crRNA) that is processed by Cas proteins to generate mature crRNA species. In type III systems, crRNAs are matured with the help of the endogenous RNase III, the Cas9 protein, and a short RNA, which is termed tracrRNA (5
). In the interference phase, the invading nucleic acid is recognized by the respective crRNA (displayed on the surface of Cas proteins) and silenced. For a detailed description of all three steps, see recent reviews (6
An essential factor for a successful interference and presumably also for the adaptation stage is the dual recognition of both the protospacer sequence and the nearby p
otif (PAM) which is found only in the natural target. This dual recognition mechanism prevents autoimmunity at the spacer encoded by the chromosomal CRISPR gene (13
). PAM sequences appear to be conserved and show a distinct relationship to the CRISPR repeat sequences, which also show significant conservation, providing a means of classification into CRISPR groups (14
). (We are using the following terms here: “CRISPR/Cas type” as classified by Makarova et al.
) that describes the whole immune system with CRISPR RNAs and the type-specific and subtype-specific Cas proteins and “CRISPR repeat clusters” or “CRISPR group” as defined by Kunin et al.
) and Mojica et al.
) that describes the classification of CRISPR groups by their repeats.) PAM sequence requirements and position vary between CRISPR/Cas types; for example, in type I systems, the PAM sequences are found directly upstream of the protospacer, whereas in CRISPR/Cas type II systems, they are located immediately downstream of the protospacer sequence (4
). Up to now, a requirement for PAM sequences by CRISPR/Cas type III systems has not been reported (4
). In the adaptation phase, PAM sequences probably play a crucial role in the selection of protospacers from the invading nucleic acid, but details of the recognition mechanism remain unclear (4
The importance of PAM sequences in the interference stage of CRISPR/Cas type I systems was recently reported by Semenova et al.
) using Escherichia coli
and by Gudbergsdottir et al.
) using Sulfolobus islandicus
. Mutation of the PAM sequence resulted in escape from interference in both organisms, showing that a correct PAM sequence is essential for target recognition by the CRISPR/Cas type I system (16
). In contrast, the CRISPR/Cas type III system of Staphylococcus epidermidis
did not require any PAM sequence for interference, suggesting that type III systems in general do not require a PAM sequence (13
PAM sequences are easily determined if spacer sequences of CRISPR loci can be matched to known virus or plasmid sequences (15
). However, it is often difficult to find matching sequences to spacers because of the limited sequence information of prokaryotic viruses and plasmids, but the current rapid expansion in whole genome, metagenomic, and metaviromic sequence studies is beginning to provide useful data even in extreme environments, such as hypersaline waters (18
Here, we provide the first insights into the function and specific roles of the CRISPR/Cas system of the halophilic euryarchaeon Haloferax volcanii. This organism possesses three CRISPR loci, one on the main chromosome and two closely spaced loci on the minichromosome pHV4 (). Between the two pHV4-encoded loci is the only Cas protein gene cassette comprising proteins belonging to CRISPR/Cas type I-B. To identify functional PAM sequences for Haloferax, we used a systematic approach using a plasmid-based invader assay.
FIGURE 1. The CRISPR/Cas system of H. volcanii. Two CRISPR genes (P1 and P2) are encoded on minichromosome pHV4 flanking the cas gene cluster. The third CRISPR gene is encoded on the chromosome (C). The cas gene cluster codes for the Cas proteins Cas1, Cas2, Cas3, (more ...)