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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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Protospacers are specifically integrated into the CRISPR locusa, Integration sites along pCRISPR. b, Magnified view of the integration sites along the ~1 kb CRISPR locus. The cyan peaks represent positions where the 3' T of the protospacer DNA was integrated whereas the black peaks represent the C 3'-OH integration events. The protospacer sequence is depicted above the plot. c, Integration sites along pUC19. d, Comparison of C 3'-OH or T 3'-OH selection in the total reads from pCRISPR and pUC19 targets (n=7,866 reads for pCRISPR and n=5,524 reads for pUC19, Chi-square test, *p<0.0001). e, Schematic of DNA cruciform formation of the repeat sequences. The orange arrows depict the cleavage sites.
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Figure 4: Protospacers are specifically integrated into the CRISPR locusa, Integration sites along pCRISPR. b, Magnified view of the integration sites along the ~1 kb CRISPR locus. The cyan peaks represent positions where the 3' T of the protospacer DNA was integrated whereas the black peaks represent the C 3'-OH integration events. The protospacer sequence is depicted above the plot. c, Integration sites along pUC19. d, Comparison of C 3'-OH or T 3'-OH selection in the total reads from pCRISPR and pUC19 targets (n=7,866 reads for pCRISPR and n=5,524 reads for pUC19, Chi-square test, *p<0.0001). e, Schematic of DNA cruciform formation of the repeat sequences. The orange arrows depict the cleavage sites.

Mentions: To determine the exact sites of protospacer integration in these reactions, we performed high-throughput sequencing of reaction products that resulted from using either pCRISPR or the parental pUC19 vector as the target of integration (Extended Data Fig. 8a). Of the 7,866 protospacer-pCRISPR junctions retrieved, ~71% mapped to the CRISPR locus (Fig. 4a and Extended Data Fig. 8b). Protospacer insertion occurred at the borders of each repeat, with the most preferred site at the first repeat adjacent to the leader (Fig. 4b). The minus strand of each repeat (the bottom strand in Fig. 4a,b that runs 5'-to-3' towards the leader sequence) is also highly preferred, highlighting the role of CRISPR repeats in providing sequence specificity for the Cas1–Cas2 complex (Fig. 4b). Sequence alignment of the integration sites revealed strong preference for sequences resembling the CRISPR repeat on both strands of pCRISPR, further supporting the selection of CRISPR repeat borders by the Cas1–Cas2 complex (Extended Data Fig. 8d-f).


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

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

Protospacers are specifically integrated into the CRISPR locusa, Integration sites along pCRISPR. b, Magnified view of the integration sites along the ~1 kb CRISPR locus. The cyan peaks represent positions where the 3' T of the protospacer DNA was integrated whereas the black peaks represent the C 3'-OH integration events. The protospacer sequence is depicted above the plot. c, Integration sites along pUC19. d, Comparison of C 3'-OH or T 3'-OH selection in the total reads from pCRISPR and pUC19 targets (n=7,866 reads for pCRISPR and n=5,524 reads for pUC19, Chi-square test, *p<0.0001). e, Schematic of DNA cruciform formation of the repeat sequences. The orange arrows depict the cleavage sites.
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Related In: Results  -  Collection

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

Figure 4: Protospacers are specifically integrated into the CRISPR locusa, Integration sites along pCRISPR. b, Magnified view of the integration sites along the ~1 kb CRISPR locus. The cyan peaks represent positions where the 3' T of the protospacer DNA was integrated whereas the black peaks represent the C 3'-OH integration events. The protospacer sequence is depicted above the plot. c, Integration sites along pUC19. d, Comparison of C 3'-OH or T 3'-OH selection in the total reads from pCRISPR and pUC19 targets (n=7,866 reads for pCRISPR and n=5,524 reads for pUC19, Chi-square test, *p<0.0001). e, Schematic of DNA cruciform formation of the repeat sequences. The orange arrows depict the cleavage sites.
Mentions: To determine the exact sites of protospacer integration in these reactions, we performed high-throughput sequencing of reaction products that resulted from using either pCRISPR or the parental pUC19 vector as the target of integration (Extended Data Fig. 8a). Of the 7,866 protospacer-pCRISPR junctions retrieved, ~71% mapped to the CRISPR locus (Fig. 4a and Extended Data Fig. 8b). Protospacer insertion occurred at the borders of each repeat, with the most preferred site at the first repeat adjacent to the leader (Fig. 4b). The minus strand of each repeat (the bottom strand in Fig. 4a,b that runs 5'-to-3' towards the leader sequence) is also highly preferred, highlighting the role of CRISPR repeats in providing sequence specificity for the Cas1–Cas2 complex (Fig. 4b). Sequence alignment of the integration sites revealed strong preference for sequences resembling the CRISPR repeat on both strands of pCRISPR, further supporting the selection of CRISPR repeat borders by the Cas1–Cas2 complex (Extended Data Fig. 8d-f).

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