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Science 19 December 2008:
Vol. 322. no. 5909, pp. 1843 - 1845
DOI: 10.1126/science.1165771
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Reports

CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Luciano A. Marraffini and Erik J. Sontheimer*

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.


Figure 1 Fig. 1. A CRISPR locus provides immunity against plasmid conjugation in S. epidermidis. (A) Organization of the RP62a CRISPR locus. Repeats and spacers (colored boxes) are followed by CRISPR-associated genes (cas1, cas2, and cas6) and cas subtype Mycobacterium tuberculosis genes (csm1 to csm6) (5). An AT-rich "leader" sequence precedes the repeat-spacer region (black box). LAM104 is an isogenic {Delta}crispr strain lacking only the repeat and spacer sequences. (B) The staphylococcal conjugative plasmid pG0400 spc1 target sequence [pG0(wt), highlighted in yellow] is shown on the top. This sequence was altered to introduce synonymous mutations to generate pG0(mut), with changes shown in red. (C) To restore interference in strain LAM104, two plasmids were introduced: pCRISPR and pCRISPR-L. (D) Conjugation was carried out by filter-mating in triplicate; the colony-forming units (CFU) per milliliter values (mean ± SD) obtained for recipients and transconjugants are shown. Recipient strains, complementing plasmids, and donor conjugative plasmids are indicated. Conjugation efficiency (Conj. Eff.) was calculated as the recipients/transconjugants ratio. [View Larger Version of this Image (40K GIF file)]
 

Figure 2 Fig. 2. CRISPR interference requires an intact target sequence in plasmid DNA but not mRNA. (A) Disruption of the pG0400 nes target sequence with the orf142-I2 self-splicing intron, generating the conjugative plasmid pG0(I2). (B) Conugation efficiency was measured as in Fig. 1D with RP62a and the {Delta}crispr mutant LAM104 as recipients for pG0(wt) and pG0(I2). To test for interference with spliced nes mRNA, RP62a and LAM104 transconjugants were also used as donors of pG0(I2) to S. epidermidis ATCC 12228. [View Larger Version of this Image (48K GIF file)]
 

Figure 3 Fig. 3. Plasmid transformation is subject to CRISPR interference. (A) Introduction of the wild-type and mutant nes target sequences (mutations highlighted in gray) into the plasmid pC194 (d, direct insertion; i, inverted insertion). The origin of replication (ori) and protein-coding genes are indicated. Stem loops denote the rep and cat transcriptional terminators. (B) RP62a and the {Delta}crispr mutant LAM104 were transformed in triplicate, with the plasmids described in (A). Transformation efficiency was calculated as CFU/µg of DNA (mean ± SD). [View Larger Version of this Image (33K GIF file)]
 

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Science. ISSN 0036-8075 (print), 1095-9203 (online)