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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

PAMs and DNA content along the E. coli BL21-AI genome(A) Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10kb. (B) DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (genbank accession NC_012947). Areas where little or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).
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Figure 7: PAMs and DNA content along the E. coli BL21-AI genome(A) Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10kb. (B) DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (genbank accession NC_012947). Areas where little or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).

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)

PAMs and DNA content along the E. coli BL21-AI genome(A) Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10kb. (B) DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (genbank accession NC_012947). Areas where little or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 7: PAMs and DNA content along the E. coli BL21-AI genome(A) Distribution of PAM (AAG) sequences. Each data point represents the number of PAMs in a window of 10kb. (B) DNA content of a culture growing in log phase. Genomic DNA was extracted from E. coli BL21-AI cells carrying the pCas plasmid, grown at log phase, and was sequenced using the Illumina technology. The resulting reads were mapped to the sequenced E. coli BL21(DE3) genome (genbank accession NC_012947). Areas where little or no reads map to the genome represent regions that are present in the reference BL21(DE3) genome but are missing from the genome of the sequenced strain (BL21-AI).
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