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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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Model of the CRISPR–Cas adaptive immunity pathway in E. coli.Mature double-stranded protospacers bearing a 3' C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5'G/3'C as the first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific recognition of the 3'-TTC-5' PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to the inability for the crRNA to recognize the strand with the 3'-TTC-5' PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly orienting the protospacer during integration.
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Figure 15: Model of the CRISPR–Cas adaptive immunity pathway in E. coli.Mature double-stranded protospacers bearing a 3' C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5'G/3'C as the first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific recognition of the 3'-TTC-5' PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to the inability for the crRNA to recognize the strand with the 3'-TTC-5' PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly orienting the protospacer during integration.

Mentions: The results presented here explain the mechanistic basis for foreign DNA acquisition during CRISPR–Cas adaptive immunity (Fig. 5). The Cas1–Cas2 complex catalyzes integration of protospacers at the leader-end of the CRISPR locus and also selects the terminal C 3'-OH as the attacking nucleophile, resulting in the 5' G on the opposite strand of the protospacer becoming the first nucleotide of the newly integrated spacer. This orientation bias, previously observed in vivo39, is a key step during immunity for productive downstream foreign DNA targeting by the Cascade complex and Cas3 effector nuclease (Extended Data Fig. 10). Interestingly, the presence of the complete AAG PAM in the protospacer is not required for in vitro integration, suggesting that a highly specific selection or processing step occurs in vivo to exclude the AA nucleotides from the mature protospacer prior to integration.


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

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

Model of the CRISPR–Cas adaptive immunity pathway in E. coli.Mature double-stranded protospacers bearing a 3' C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5'G/3'C as the first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific recognition of the 3'-TTC-5' PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to the inability for the crRNA to recognize the strand with the 3'-TTC-5' PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly orienting the protospacer during integration.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 15: Model of the CRISPR–Cas adaptive immunity pathway in E. coli.Mature double-stranded protospacers bearing a 3' C-OH are site-specifically integrated into the leader-end of the CRISPR locus. Correct protospacer integration (left) results in the 5'G/3'C as the first nucleotide of the spacer, proximal to the leader. After transcription of the CRISPR locus and subsequent crRNA processing, foreign DNA destruction is initiated by strand-specific recognition of the 3'-TTC-5' PAM sequence in the target strand by the crRNA-guided Cascade complex. Incorrect protospacer integration (right) cannot initiate foreign DNA destruction due to the inability for the crRNA to recognize the strand with the 3'-TTC-5' PAM. Thus, foreign DNA interference during CRISPR–Cas adaptive immunity relies on the Cas1–Cas2 complex for correctly orienting the protospacer during integration.
Mentions: The results presented here explain the mechanistic basis for foreign DNA acquisition during CRISPR–Cas adaptive immunity (Fig. 5). The Cas1–Cas2 complex catalyzes integration of protospacers at the leader-end of the CRISPR locus and also selects the terminal C 3'-OH as the attacking nucleophile, resulting in the 5' G on the opposite strand of the protospacer becoming the first nucleotide of the newly integrated spacer. This orientation bias, previously observed in vivo39, is a key step during immunity for productive downstream foreign DNA targeting by the Cascade complex and Cas3 effector nuclease (Extended Data Fig. 10). Interestingly, the presence of the complete AAG PAM in the protospacer is not required for in vitro integration, suggesting that a highly specific selection or processing step occurs in vivo to exclude the AA nucleotides from the mature protospacer prior to integration.

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