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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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Related in: MedlinePlus

A pre-existing spacer:protospacer match accelerates Cas-dependent plasmid loss. (A) P. atrosepticum contains a Type I-F CRISPR-Cas system composed of three CRISPR arrays (1–3; gray arrows) and an operon of 6 cas genes (colored arrows). CRISPR2 consists of 10 spacers and spacer 6 (from leader proximal end; blue) perfectly matches a protospacer (red) in eca0560 in the chromosomal island HAI2, but has a TG PAM variant. (B) Schematic of the plasmid loss assays. P. atrosepticum ΔHAI2 carrying pTRB30 (control) or pPF189 (eca0560 primed; depicted) plasmids were grown without selection for 5 days and plasmid loss was scored by replica-plating on non-selective (NS) and selective (S) media. (C) Plasmid loss of a control plasmid (pTRB30) and the primed plasmid (pPF189) over 5 days when cultured in ΔHAI2 or ΔHAI2Δcas backgrounds. Data shown are the mean ± SD of three biological replicates.
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Figure 1: A pre-existing spacer:protospacer match accelerates Cas-dependent plasmid loss. (A) P. atrosepticum contains a Type I-F CRISPR-Cas system composed of three CRISPR arrays (1–3; gray arrows) and an operon of 6 cas genes (colored arrows). CRISPR2 consists of 10 spacers and spacer 6 (from leader proximal end; blue) perfectly matches a protospacer (red) in eca0560 in the chromosomal island HAI2, but has a TG PAM variant. (B) Schematic of the plasmid loss assays. P. atrosepticum ΔHAI2 carrying pTRB30 (control) or pPF189 (eca0560 primed; depicted) plasmids were grown without selection for 5 days and plasmid loss was scored by replica-plating on non-selective (NS) and selective (S) media. (C) Plasmid loss of a control plasmid (pTRB30) and the primed plasmid (pPF189) over 5 days when cultured in ΔHAI2 or ΔHAI2Δcas backgrounds. Data shown are the mean ± SD of three biological replicates.

Mentions: Five millilitres cultures of P. atrosepticum ΔHAI2 with pTRB30 (vector control) or pPF189 were grown overnight without antibiotic selection (Figure 1). Note that 10 μl were used to inoculate a fresh overnight culture and dilutions were plated onto LBA. This was repeated over 5 days and performed in triplicate. Colonies (100) from each replicate were patched onto LBA ± Km. Km sensitive (KmS) colonies were screened by PCR for new spacers as described later.


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)

A pre-existing spacer:protospacer match accelerates Cas-dependent plasmid loss. (A) P. atrosepticum contains a Type I-F CRISPR-Cas system composed of three CRISPR arrays (1–3; gray arrows) and an operon of 6 cas genes (colored arrows). CRISPR2 consists of 10 spacers and spacer 6 (from leader proximal end; blue) perfectly matches a protospacer (red) in eca0560 in the chromosomal island HAI2, but has a TG PAM variant. (B) Schematic of the plasmid loss assays. P. atrosepticum ΔHAI2 carrying pTRB30 (control) or pPF189 (eca0560 primed; depicted) plasmids were grown without selection for 5 days and plasmid loss was scored by replica-plating on non-selective (NS) and selective (S) media. (C) Plasmid loss of a control plasmid (pTRB30) and the primed plasmid (pPF189) over 5 days when cultured in ΔHAI2 or ΔHAI2Δcas backgrounds. Data shown are the mean ± SD of three biological replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: A pre-existing spacer:protospacer match accelerates Cas-dependent plasmid loss. (A) P. atrosepticum contains a Type I-F CRISPR-Cas system composed of three CRISPR arrays (1–3; gray arrows) and an operon of 6 cas genes (colored arrows). CRISPR2 consists of 10 spacers and spacer 6 (from leader proximal end; blue) perfectly matches a protospacer (red) in eca0560 in the chromosomal island HAI2, but has a TG PAM variant. (B) Schematic of the plasmid loss assays. P. atrosepticum ΔHAI2 carrying pTRB30 (control) or pPF189 (eca0560 primed; depicted) plasmids were grown without selection for 5 days and plasmid loss was scored by replica-plating on non-selective (NS) and selective (S) media. (C) Plasmid loss of a control plasmid (pTRB30) and the primed plasmid (pPF189) over 5 days when cultured in ΔHAI2 or ΔHAI2Δcas backgrounds. Data shown are the mean ± SD of three biological replicates.
Mentions: Five millilitres cultures of P. atrosepticum ΔHAI2 with pTRB30 (vector control) or pPF189 were grown overnight without antibiotic selection (Figure 1). Note that 10 μl were used to inoculate a fresh overnight culture and dilutions were plated onto LBA. This was repeated over 5 days and performed in triplicate. Colonies (100) from each replicate were patched onto LBA ± Km. Km sensitive (KmS) colonies were screened by PCR for new spacers as described later.

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