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CRISPR adaptation biases explain preference for acquisition of foreign DNA.

Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R - Nature (2015)

Bottom Line: CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids.In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array.We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

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Related in: MedlinePlus

Distribution of protospacers across (A) pCtrl-Chi and (B) pChi plasmidsCircular representation of the 4.7kb plasmid is presented, with the inserted 4-Chi cluster present at the top-middle of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar indicates 100k spacers. Pooled protospacers from two replicates are presented for each panel.
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Figure 11: Distribution of protospacers across (A) pCtrl-Chi and (B) pChi plasmidsCircular representation of the 4.7kb plasmid is presented, with the inserted 4-Chi cluster present at the top-middle of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar indicates 100k spacers. Pooled protospacers from two replicates are presented for each panel.

Mentions: The involvement of Chi sites, as points where spacer acquisition activity is terminated, provides another axis for the avoidance of self DNA in CRISPR adaptation. Since the pCas plasmid is completely devoid of Chi sites, its DNA will be fully degraded by RecBCD following any dsDNA break, providing plenty of potential substrate for Cas1+2. In contrast, the high density of Chi sites on the bacterial chromosome serves for the relative avoidance of Cas1+2 to acquire spacers from the chromosome, because RecBCD will only degrade the chromosomal DNA until reaching the nearest Chi site (Fig. 5a-b). Indeed, the ~10 fold higher acquisition frequency from the self choromosome seen in the recB, recC and recD deletion strains conforms with the natural 14-fold enrichment of Chi sites on the chromosome. To further examine whether Chi sites limit spacer acquisition, we performed spacer acquisition experiments with a plasmid that was engineered to contain a cluster of 4 consecutive Chi sites. As expected, an increased preference for chrmomosomal DNA in spacer acquisition was measured for the Chi-containing plasmid (Fig 4g; Extended Data Table 3b; Extended Data Fig. 6).


CRISPR adaptation biases explain preference for acquisition of foreign DNA.

Levy A, Goren MG, Yosef I, Auster O, Manor M, Amitai G, Edgar R, Qimron U, Sorek R - Nature (2015)

Distribution of protospacers across (A) pCtrl-Chi and (B) pChi plasmidsCircular representation of the 4.7kb plasmid is presented, with the inserted 4-Chi cluster present at the top-middle of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar indicates 100k spacers. Pooled protospacers from two replicates are presented for each panel.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561520&req=5

Figure 11: Distribution of protospacers across (A) pCtrl-Chi and (B) pChi plasmidsCircular representation of the 4.7kb plasmid is presented, with the inserted 4-Chi cluster present at the top-middle of the circle. Black bars indicate the number of PAM-derived spacers sequenced from each position; green bars represent non-PAM spacers. Scale bar indicates 100k spacers. Pooled protospacers from two replicates are presented for each panel.
Mentions: The involvement of Chi sites, as points where spacer acquisition activity is terminated, provides another axis for the avoidance of self DNA in CRISPR adaptation. Since the pCas plasmid is completely devoid of Chi sites, its DNA will be fully degraded by RecBCD following any dsDNA break, providing plenty of potential substrate for Cas1+2. In contrast, the high density of Chi sites on the bacterial chromosome serves for the relative avoidance of Cas1+2 to acquire spacers from the chromosome, because RecBCD will only degrade the chromosomal DNA until reaching the nearest Chi site (Fig. 5a-b). Indeed, the ~10 fold higher acquisition frequency from the self choromosome seen in the recB, recC and recD deletion strains conforms with the natural 14-fold enrichment of Chi sites on the chromosome. To further examine whether Chi sites limit spacer acquisition, we performed spacer acquisition experiments with a plasmid that was engineered to contain a cluster of 4 consecutive Chi sites. As expected, an increased preference for chrmomosomal DNA in spacer acquisition was measured for the Chi-containing plasmid (Fig 4g; Extended Data Table 3b; Extended Data Fig. 6).

Bottom Line: CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids.In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array.We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

ABSTRACT
CRISPR-Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA ('spacers') are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages.

Show MeSH
Related in: MedlinePlus