Limits...
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.

Show MeSH

Related in: MedlinePlus

Cas1–Cas2 correctly orients the protospacer DNA during integrationMapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide ends (a) “wild type” 3' C and 3' T, (c) 3' A and 3' T, and (e) 3' C and 3' C. The red arrow in c and e points to the nucleotide change in the protospacer DNA compared to the “wild type” sequence in a. The protospacer DNA 3' nucleotide and the CRISPR locus strand biases in a, c, e are plotted in b, d and f, respectively, as percentages of integration events within the CRISPR locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and *p<0.0001 by Chi-square test. The n values for b, d and f are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359072&req=5

Figure 14: Cas1–Cas2 correctly orients the protospacer DNA during integrationMapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide ends (a) “wild type” 3' C and 3' T, (c) 3' A and 3' T, and (e) 3' C and 3' C. The red arrow in c and e points to the nucleotide change in the protospacer DNA compared to the “wild type” sequence in a. The protospacer DNA 3' nucleotide and the CRISPR locus strand biases in a, c, e are plotted in b, d and f, respectively, as percentages of integration events within the CRISPR locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and *p<0.0001 by Chi-square test. The n values for b, d and f are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively.

Mentions: In E. coli, newly acquired spacers harbor a 5' G as the first nucleotide flanking the leader-proximal end of the repeats, which originates from the last nucleotide of the AAG protospacer-adjacent motif (PAM) from foreign DNA13-15,37-39. Such positional specificity is critical for crRNA-guided interference, as a mutation in this position of the corresponding crRNA disrupts PAM binding and subsequent target destruction40-42. We found that ~73% of all integration events into pCRISPR utilized the 3' C end instead of the 3' T end of protospacer DNA during integration (see Fig. 4b for protospacer sequence), and there was a strong preference for this nucleotide to attack the minus strand of the repeat sequence (Fig. 4b,d,e). A similar nucleotide bias was observed in the pUC19 target plasmid sequence data (Fig. 4d). This preference positions the G at the 5' end of the protospacer substrate as the first nucleotide of the newly integrated spacer in the CRISPR locus (Fig. 5). When we used protospacer DNAs lacking a 3' C or bearing 3' C on both ends, the preference for integration into the minus strand of the CRISPR locus was significantly decreased (Extended Data Fig. 9). Thus, the Cas1–Cas2 complex plays a critical role in correctly orienting the C 3'-OH end of protospacer DNA substrates for incorporation within the CRISPR locus.


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

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

Cas1–Cas2 correctly orients the protospacer DNA during integrationMapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide ends (a) “wild type” 3' C and 3' T, (c) 3' A and 3' T, and (e) 3' C and 3' C. The red arrow in c and e points to the nucleotide change in the protospacer DNA compared to the “wild type” sequence in a. The protospacer DNA 3' nucleotide and the CRISPR locus strand biases in a, c, e are plotted in b, d and f, respectively, as percentages of integration events within the CRISPR locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and *p<0.0001 by Chi-square test. The n values for b, d and f are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 14: Cas1–Cas2 correctly orients the protospacer DNA during integrationMapped integration sites along the CRISPR locus of pCRISPR when using protospacer DNA with nucleotide ends (a) “wild type” 3' C and 3' T, (c) 3' A and 3' T, and (e) 3' C and 3' C. The red arrow in c and e points to the nucleotide change in the protospacer DNA compared to the “wild type” sequence in a. The protospacer DNA 3' nucleotide and the CRISPR locus strand biases in a, c, e are plotted in b, d and f, respectively, as percentages of integration events within the CRISPR locus. The black and clear bars represent the (−) and (+) strands of the CRISPR locus, respectively. NS corresponds to not significant and *p<0.0001 by Chi-square test. The n values for b, d and f are 5,623, 5,685 and 12,453 reads along the CRISPR locus, respectively.
Mentions: In E. coli, newly acquired spacers harbor a 5' G as the first nucleotide flanking the leader-proximal end of the repeats, which originates from the last nucleotide of the AAG protospacer-adjacent motif (PAM) from foreign DNA13-15,37-39. Such positional specificity is critical for crRNA-guided interference, as a mutation in this position of the corresponding crRNA disrupts PAM binding and subsequent target destruction40-42. We found that ~73% of all integration events into pCRISPR utilized the 3' C end instead of the 3' T end of protospacer DNA during integration (see Fig. 4b for protospacer sequence), and there was a strong preference for this nucleotide to attack the minus strand of the repeat sequence (Fig. 4b,d,e). A similar nucleotide bias was observed in the pUC19 target plasmid sequence data (Fig. 4d). This preference positions the G at the 5' end of the protospacer substrate as the first nucleotide of the newly integrated spacer in the CRISPR locus (Fig. 5). When we used protospacer DNAs lacking a 3' C or bearing 3' C on both ends, the preference for integration into the minus strand of the CRISPR locus was significantly decreased (Extended Data Fig. 9). Thus, the Cas1–Cas2 complex plays a critical role in correctly orienting the C 3'-OH end of protospacer DNA substrates for incorporation within the CRISPR locus.

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