Protospacer recognition motifs: mixed identities and functional diversity

Abstract

Protospacer adjacent motifs (PAMs) were originally characterized for CRISPR-Cas systems that were classified on the basis of their CRISPR repeat sequences. A few short 2-5 bp sequences were identified adjacent to one end of the protospacers. Experimental and bioinformatical results linked the motif to the excision of protospacers and their insertion into CRISPR loci. Subsequently, evidence accumulated from different virus- and plasmid-targeting assays, suggesting that these motifs were also recognized during DNA interference, at least for the recently classified type I and type II CRISPR-based systems. The two processes, spacer acquisition and protospacer interference, employ different molecular mechanisms, and there is increasing evidence to suggest that the sequence motifs that are recognized, while overlapping, are unlikely to be identical. In this article, we consider the properties of PAM sequences and summarize the evidence for their dual functional roles. It is proposed to use the terms protospacer associated motif (PAM) for the conserved DNA sequence and to employ spacer acqusition motif (SAM) and target interference motif (TIM), respectively, for acquisition and interference recognition sites.

Keywords: CRISPR; PAM; SAM; TIM; adaptive immunity; protospacer.

Figure 1. Coevolution of sequences of CRISPR repeats, Cas1 proteins and PAMs, for members of the Sulfolobales. The type I CRISPR systems fall into three main subtypes, I-A, I-D and I-B. The I-A systems are the most common and can be classified into distinct subfamilies I-A₁ and I-A₂. (A) A neighbor joining tree of CRISPR repeat sequences (left) is juxtaposed with that of the translated sequences of cas1 genes (right). CRISPR loci are identified by the short name of the organism and the number of CRISPR repeats. cas1 genes are colored according to the CRISPR subtypes (I-A₁, blue; I-A₂, pink; I-D, yellow and I-B, green). (B) Protospacer matches from spacers of the four distinct subtypes of CRISPR arrays yield dominant consensus PAMs, CCN for I-A₁, TCN for I-A₂, GTN for I-D and with ATTA for two protospacers predicted for subtype I-B. Motifs were derived from spacer-protospacer matches on viral or plasmid genomes of the Sulfolobales exhibiting five or less mismatches. The total number of predicted protospacers is given in brackets.

Figure 2. In silico determination of multiple spacer matches to the bicaudavirus ATV in three CRISPR loci of the crenarchaeon M. sedula. Repeat-spacer units from sections of type I-A₁ CRISPR arrays are depicted as arrowheads, directed away from the leader. Numbers to the left and right delineate the range of repeat-spacer units depicted. Shaded units yield close matches (≤ 5 nt mismatches) to protospacers in ATV. The orientation of the matching spacers with respect to the ATV genome is indicated by a dot above or below the shaded arrowheads.

Figure 3. Overview of putative PAM, SAM and TIM interactions during acquisition and interference in type I and type II CRISPR systems. (A) The spacer acquisition motif (SAM) is recognized on the invader DNA by the Cas protein acquisition complex, which leads to the protospacer being excised by a putative ruler mechanism and reinserted into a CRISPR locus by another putative ruler mechanism. During interference by type I systems the target interference motif (TIM), on the crRNA-complementary DNA strand, is recognized by the Cas protein-crRNA complex where both TIM recognition and crRNA annealing are required for successful invader cleavage. (B) In type II systems, the SAM/PAM motif is inferred to be recognized by a mechanism related to the type I system but inverted on the dsDNA whereas TIM recognition occurs on the non-complementary DNA strand to the crRNA.