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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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Chromosome-scale hotspots for spacer acquisition(A) Distribution of protospacers across the E. coli BL21-AI genome. Protospacers were deduced from aligning new spacers, acquired into the CRISPR I array after 16 hours growth with no arabinose, to the bacterial genome. Only unique protospacers are presented, to avoid possible biases stemming from PCR amplification of the CRISPR array. Pooled protospacers from two replicates are presented. (B) Protospacer density across a circular representation of the E. coli genome, normalized to the DNA content of the culture. Dark brown, normalized protospacer numbers; orange, PAM density. (C) Protospacer distribution at the Ter region. Protospacer density is shown in 1kb windows. (D) Protospacer density in an E. coli BL21-AI in which the native 23bp-long TerB site was engineered into the pheA locus.
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Figure 1: Chromosome-scale hotspots for spacer acquisition(A) Distribution of protospacers across the E. coli BL21-AI genome. Protospacers were deduced from aligning new spacers, acquired into the CRISPR I array after 16 hours growth with no arabinose, to the bacterial genome. Only unique protospacers are presented, to avoid possible biases stemming from PCR amplification of the CRISPR array. Pooled protospacers from two replicates are presented. (B) Protospacer density across a circular representation of the E. coli genome, normalized to the DNA content of the culture. Dark brown, normalized protospacer numbers; orange, PAM density. (C) Protospacer distribution at the Ter region. Protospacer density is shown in 1kb windows. (D) Protospacer density in an E. coli BL21-AI in which the native 23bp-long TerB site was engineered into the pheA locus.

Mentions: Although only a small minority of spacers was derived from the E. coli chromosome, the extensive number of sequenced spacers allowed us to examine chromosome-scale patterns of spacer acquisition. Remarkably, strong biases in spacer acquisition were observed, defining several protospacer hotspots (Fig. 1a). As the protospacer adjacent motif (PAM) density on the chromosome scale is largely uniform (Fig. 1b, Extended Data Fig. 2), these protospacer hotspots could not be explained by excessive localization of PAM sequences in specific areas of the genome. We further investigated each of the hotspots in search for a mechanism that would explain the observed biases.


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)

Chromosome-scale hotspots for spacer acquisition(A) Distribution of protospacers across the E. coli BL21-AI genome. Protospacers were deduced from aligning new spacers, acquired into the CRISPR I array after 16 hours growth with no arabinose, to the bacterial genome. Only unique protospacers are presented, to avoid possible biases stemming from PCR amplification of the CRISPR array. Pooled protospacers from two replicates are presented. (B) Protospacer density across a circular representation of the E. coli genome, normalized to the DNA content of the culture. Dark brown, normalized protospacer numbers; orange, PAM density. (C) Protospacer distribution at the Ter region. Protospacer density is shown in 1kb windows. (D) Protospacer density in an E. coli BL21-AI in which the native 23bp-long TerB site was engineered into the pheA locus.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 1: Chromosome-scale hotspots for spacer acquisition(A) Distribution of protospacers across the E. coli BL21-AI genome. Protospacers were deduced from aligning new spacers, acquired into the CRISPR I array after 16 hours growth with no arabinose, to the bacterial genome. Only unique protospacers are presented, to avoid possible biases stemming from PCR amplification of the CRISPR array. Pooled protospacers from two replicates are presented. (B) Protospacer density across a circular representation of the E. coli genome, normalized to the DNA content of the culture. Dark brown, normalized protospacer numbers; orange, PAM density. (C) Protospacer distribution at the Ter region. Protospacer density is shown in 1kb windows. (D) Protospacer density in an E. coli BL21-AI in which the native 23bp-long TerB site was engineered into the pheA locus.
Mentions: Although only a small minority of spacers was derived from the E. coli chromosome, the extensive number of sequenced spacers allowed us to examine chromosome-scale patterns of spacer acquisition. Remarkably, strong biases in spacer acquisition were observed, defining several protospacer hotspots (Fig. 1a). As the protospacer adjacent motif (PAM) density on the chromosome scale is largely uniform (Fig. 1b, Extended Data Fig. 2), these protospacer hotspots could not be explained by excessive localization of PAM sequences in specific areas of the genome. We further investigated each of the hotspots in search for a mechanism that would explain the observed biases.

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