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Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

Richter C, Dy RL, McKenzie RE, Watson BN, Taylor C, Chang JT, McNeil MB, Staals RH, Fineran PC - Nucleic Acids Res. (2014)

Bottom Line: Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems.Endogenous expression of the cas genes was sufficient, yet required, for priming.Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.

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The Type I-F PAM. (A) Schematic of the crRNA bound to the protospacer showing the location of the PAM. (B) Spacer length distribution. (C) Sequence Logo of 8 nt of 5′ and 3′ protospacer flanking sequences. The consensus –1/–2 GG PAM is shown. Number of GG dinucleotides at each position for (D) all protospacers and (E) protospacers lacking the consensus –1/–2 GG grouped according to spacer length. In (D) the inset shows the GG dinucleotides for the 31 and 34 nt spacers.
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Figure 6: The Type I-F PAM. (A) Schematic of the crRNA bound to the protospacer showing the location of the PAM. (B) Spacer length distribution. (C) Sequence Logo of 8 nt of 5′ and 3′ protospacer flanking sequences. The consensus –1/–2 GG PAM is shown. Number of GG dinucleotides at each position for (D) all protospacers and (E) protospacers lacking the consensus –1/–2 GG grouped according to spacer length. In (D) the inset shows the GG dinucleotides for the 31 and 34 nt spacers.

Mentions: The Type I-F system of P. atrosepticum contains CRISPR-4 (cluster 4) repeats (34), which were proposed in a bioinformatic study to possess a 5′-protospacer-GG-3′ PAM (21) (Figure 6A). However, no study has analyzed Type I-F PAMs from a large number of experimental acquisition events. Therefore, we combined our data to determine the Type I-F PAM for new spacers incorporated through priming (n = 351). Of the new spacers, 87% were 32 nt, 12% were 33 nt, <1% were 34 nt and one case of 31 nt was detected (Figure 6B). The 5′ and 3′ flanks of all protospacers were aligned and sequence logos generated (Figure 6C). No conservation was detected in the 5′ flanking sequence or in the protospacer (Supplementary Figure S5), but a GG PAM was detected in the –1 and –2 positions of the 3′ flanking sequence (i.e. a 5′-protospacer-GG-3′ PAM). When protospacer flanks were analyzed in separate groups based on length, the 32 and 33 nt groups displayed the same overall GG PAM (Figure 6D and Supplementary Figure S5A and B).


Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

Richter C, Dy RL, McKenzie RE, Watson BN, Taylor C, Chang JT, McNeil MB, Staals RH, Fineran PC - Nucleic Acids Res. (2014)

The Type I-F PAM. (A) Schematic of the crRNA bound to the protospacer showing the location of the PAM. (B) Spacer length distribution. (C) Sequence Logo of 8 nt of 5′ and 3′ protospacer flanking sequences. The consensus –1/–2 GG PAM is shown. Number of GG dinucleotides at each position for (D) all protospacers and (E) protospacers lacking the consensus –1/–2 GG grouped according to spacer length. In (D) the inset shows the GG dinucleotides for the 31 and 34 nt spacers.
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Figure 6: The Type I-F PAM. (A) Schematic of the crRNA bound to the protospacer showing the location of the PAM. (B) Spacer length distribution. (C) Sequence Logo of 8 nt of 5′ and 3′ protospacer flanking sequences. The consensus –1/–2 GG PAM is shown. Number of GG dinucleotides at each position for (D) all protospacers and (E) protospacers lacking the consensus –1/–2 GG grouped according to spacer length. In (D) the inset shows the GG dinucleotides for the 31 and 34 nt spacers.
Mentions: The Type I-F system of P. atrosepticum contains CRISPR-4 (cluster 4) repeats (34), which were proposed in a bioinformatic study to possess a 5′-protospacer-GG-3′ PAM (21) (Figure 6A). However, no study has analyzed Type I-F PAMs from a large number of experimental acquisition events. Therefore, we combined our data to determine the Type I-F PAM for new spacers incorporated through priming (n = 351). Of the new spacers, 87% were 32 nt, 12% were 33 nt, <1% were 34 nt and one case of 31 nt was detected (Figure 6B). The 5′ and 3′ flanks of all protospacers were aligned and sequence logos generated (Figure 6C). No conservation was detected in the 5′ flanking sequence or in the protospacer (Supplementary Figure S5), but a GG PAM was detected in the –1 and –2 positions of the 3′ flanking sequence (i.e. a 5′-protospacer-GG-3′ PAM). When protospacer flanks were analyzed in separate groups based on length, the 32 and 33 nt groups displayed the same overall GG PAM (Figure 6D and Supplementary Figure S5A and B).

Bottom Line: Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems.Endogenous expression of the cas genes was sufficient, yet required, for priming.Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.

Show MeSH
Related in: MedlinePlus