Short motif sequences determine the targets of the prokaryotic CRISPR defence system.
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated CRISPR-associated sequence (CAS) proteins constitute a novel antiviral defence system that is widespread in prokaryotes. Repeats are separated by spacers, some of them homologous to sequences in mobile genetic elements. Although the whole process involved remains uncharacterized, it is known that new spacers are incorporated into CRISPR loci of the host during a phage challenge, conferring specific resistance against the virus. Moreover, it has been demonstrated that such interference is based on small RNAs carrying a spacer. These RNAs would guide the defence apparatus to foreign molecules carrying sequences that match the spacers. Despite this essential role, the spacer uptake mechanism has not been addressed. A first step forward came from the detection of motifs associated with spacer precursors (proto-spacers) of Streptococcus thermophilus, revealing a specific recognition of donor sequences in this species. Here we show that the conservation of proto-spacer adjacent motifs (PAMs) is a common theme for the most diverse CRISPR systems. The PAM sequence depends on the CRISPR-CAS variant, implying that there is a CRISPR-type-specific (motif-directed) choice of the spacers, which subsequently determines the interference target. PAMs also direct the orientation of spacers in the repeat arrays. Remarkably, observations based on such polarity argue against a recognition of the spacer precursors on transcript RNA molecules as a general rule.
Mojica
F J M
FJM
Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, E-03080 Alicante, Spain.
Díez-Villaseñor
C
C
Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, E-03080 Alicante, Spain.
García-Martínez
J
J
Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, E-03080 Alicante, Spain.
Almendros
C
C
Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, E-03080 Alicante, Spain.
D017124:Conserved Sequence; D021901:DNA, Intergenic / Q000235:genetics*; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D048249:Streptococcus thermophilus / Q000235:genetics*
D021901:DNA, Intergenic; D012329:RNA, Bacterial