Limits...
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.Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays.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

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

License
getmorefigures.php?uid=PMC4117759&req=5

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

License
Show All Figures
getmorefigures.php?uid=PMC4117759&req=5

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.Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays.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