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Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nuñez JK, Lee AS, Engelman A, Doudna JA - Nature (2015)

Bottom Line: Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases.Cas1 is the catalytic subunit and Cas2 substantially increases integration activity.Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.

ABSTRACT
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

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Cas1–Cas2 can integrate various lengths of double-stranded DNA with blunt- or 3'-overhang ends into a supercoiled target plasmida, Integration assays using the indicated lengths of protospacer DNA. b, Integration assays using varying 5' or 3' overhang lengths. c,d, A comparison of integration assays using pCRISPR or Nb.BbvCI-nicked pCRISPR target. e, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the cyan arrows correspond to the Band X product. The data presented in a-e are representative of at least three replicates.
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Figure 11: Cas1–Cas2 can integrate various lengths of double-stranded DNA with blunt- or 3'-overhang ends into a supercoiled target plasmida, Integration assays using the indicated lengths of protospacer DNA. b, Integration assays using varying 5' or 3' overhang lengths. c,d, A comparison of integration assays using pCRISPR or Nb.BbvCI-nicked pCRISPR target. e, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the cyan arrows correspond to the Band X product. The data presented in a-e are representative of at least three replicates.

Mentions: We next investigated the DNA protospacer and target DNA requirements for integration. Single-stranded protospacer DNA failed to support the reaction (Fig. 3a, b). The Cas1–Cas2 complex accommodated various protospacer lengths in vitro despite the strict 33 bp requirement for spacer acquisition in vivo (Extended Data Fig. 6a), suggesting that protospacer length is pre-determined before integration in vivo by an unknown mechanism. The Cas1–Cas2 complex integrated DNA substrates with blunt-ends or with 3'-overhangs up to 5 nt in length (Extended Data Fig. 6b). In contrast to retroviral integrases31, substrates with 5'-overhangs were nonviable (Extended Data Fig. 6b).


Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nuñez JK, Lee AS, Engelman A, Doudna JA - Nature (2015)

Cas1–Cas2 can integrate various lengths of double-stranded DNA with blunt- or 3'-overhang ends into a supercoiled target plasmida, Integration assays using the indicated lengths of protospacer DNA. b, Integration assays using varying 5' or 3' overhang lengths. c,d, A comparison of integration assays using pCRISPR or Nb.BbvCI-nicked pCRISPR target. e, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the cyan arrows correspond to the Band X product. The data presented in a-e are representative of at least three replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359072&req=5

Figure 11: Cas1–Cas2 can integrate various lengths of double-stranded DNA with blunt- or 3'-overhang ends into a supercoiled target plasmida, Integration assays using the indicated lengths of protospacer DNA. b, Integration assays using varying 5' or 3' overhang lengths. c,d, A comparison of integration assays using pCRISPR or Nb.BbvCI-nicked pCRISPR target. e, Integration assay using different target plasmids with or without a CRISPR locus. The green arrows correspond to the relaxed product of each target and the cyan arrows correspond to the Band X product. The data presented in a-e are representative of at least three replicates.
Mentions: We next investigated the DNA protospacer and target DNA requirements for integration. Single-stranded protospacer DNA failed to support the reaction (Fig. 3a, b). The Cas1–Cas2 complex accommodated various protospacer lengths in vitro despite the strict 33 bp requirement for spacer acquisition in vivo (Extended Data Fig. 6a), suggesting that protospacer length is pre-determined before integration in vivo by an unknown mechanism. The Cas1–Cas2 complex integrated DNA substrates with blunt-ends or with 3'-overhangs up to 5 nt in length (Extended Data Fig. 6b). In contrast to retroviral integrases31, substrates with 5'-overhangs were nonviable (Extended Data Fig. 6b).

Bottom Line: Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases.Cas1 is the catalytic subunit and Cas2 substantially increases integration activity.Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.

ABSTRACT
Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

Show MeSH
Related in: MedlinePlus