Limits...
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.

Show MeSH

Related in: MedlinePlus

Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns(A) The overall number of protospacers around all Chi sites in E. coli BL21-AI, that are not included in the CRISPR region (950,000-1,050,000) or the Ter region (2M-2.5M), is shown in windows of 0.5 kb. (B) Protospacer hotspot peak resulting from a dsDNA break formed by the homing endonuclease I-SceI.(C) The overall number of protospacers around all Chi sites that are not included in the CRISPR or the Ter regions in a BL21-AIΔrecB strain. (D) The protospacer hotspot at the CRISPR region in the BL21-AIΔrecB strain is not confined by a Chi site (compare to the same hotspot in the WT strain, Fig. 3A). (E) Adaption levels in WT BL21-AI and BL21-AIΔrecB, ΔrecC or ΔrecD strains following overnight growth without arabinose induction of Cas1+2. (F) Percent new spacers derived from the self chromosome in the experiment described in Panel E. (G) Percent new spacers derived from the self chromosome in the presence of a plasmid that contains a cluster of 4 Chi sites (pChi) as compared to an identical plasmid that lacks Chi sites (pCtrl-Chi). For panels E-G, average and error margins for two biological replicates are shown.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561520&req=5

Figure 4: Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns(A) The overall number of protospacers around all Chi sites in E. coli BL21-AI, that are not included in the CRISPR region (950,000-1,050,000) or the Ter region (2M-2.5M), is shown in windows of 0.5 kb. (B) Protospacer hotspot peak resulting from a dsDNA break formed by the homing endonuclease I-SceI.(C) The overall number of protospacers around all Chi sites that are not included in the CRISPR or the Ter regions in a BL21-AIΔrecB strain. (D) The protospacer hotspot at the CRISPR region in the BL21-AIΔrecB strain is not confined by a Chi site (compare to the same hotspot in the WT strain, Fig. 3A). (E) Adaption levels in WT BL21-AI and BL21-AIΔrecB, ΔrecC or ΔrecD strains following overnight growth without arabinose induction of Cas1+2. (F) Percent new spacers derived from the self chromosome in the experiment described in Panel E. (G) Percent new spacers derived from the self chromosome in the presence of a plasmid that contains a cluster of 4 Chi sites (pChi) as compared to an identical plasmid that lacks Chi sites (pCtrl-Chi). For panels E-G, average and error margins for two biological replicates are shown.

Mentions: Several lines of evidence support this hypothesis. First, the orientation of the Chi sites at the protospacer peaks was always consistent with the dsDNA break occurring at the fork direction rather than the other side, and the first properly oriented Chi site upstream to the stalled fork was always the site of peak boundary (Fig. 3a-d). Second, even outside the strong protospacer hotspots, there was a significant asymmetry in protospacer density upstream and downstream Chi sites (Fig. 4a). The effect of this asymmetry was seen up to a distance of about 5-10kb from the Chi site, consistent with an average distance of ~5kb between Chi sites in the E. coli genome 22. Third, inducing a single, site specific dsDNA break in the chromosome using the homing endonuclease I-SceI resulted in a clear protospacer hotspot that peaked at the site of the dsDNA break and was confined by Chi sites in the proper orientations (Fig. 4b), directly linking dsDNA breaks to spacer acquisition hotspots. Fourth, co-immunoprecipitation assays suggested that Cas1 interacts with RecB and RecC 27 (although these interactions were not verified using purified proteins), supporting a model where the Cas1+2 complex is directly fed from RecBCD DNA degradation products. And finally, Cas1 was shown to efficiently bind ssDNA, which is amply generated during RecBCD DNA processing activity 23,27.


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)

Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns(A) The overall number of protospacers around all Chi sites in E. coli BL21-AI, that are not included in the CRISPR region (950,000-1,050,000) or the Ter region (2M-2.5M), is shown in windows of 0.5 kb. (B) Protospacer hotspot peak resulting from a dsDNA break formed by the homing endonuclease I-SceI.(C) The overall number of protospacers around all Chi sites that are not included in the CRISPR or the Ter regions in a BL21-AIΔrecB strain. (D) The protospacer hotspot at the CRISPR region in the BL21-AIΔrecB strain is not confined by a Chi site (compare to the same hotspot in the WT strain, Fig. 3A). (E) Adaption levels in WT BL21-AI and BL21-AIΔrecB, ΔrecC or ΔrecD strains following overnight growth without arabinose induction of Cas1+2. (F) Percent new spacers derived from the self chromosome in the experiment described in Panel E. (G) Percent new spacers derived from the self chromosome in the presence of a plasmid that contains a cluster of 4 Chi sites (pChi) as compared to an identical plasmid that lacks Chi sites (pCtrl-Chi). For panels E-G, average and error margins for two biological replicates are shown.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 4: Involvement of the dsDNA break repair machinery in defining spacer acquisition patterns(A) The overall number of protospacers around all Chi sites in E. coli BL21-AI, that are not included in the CRISPR region (950,000-1,050,000) or the Ter region (2M-2.5M), is shown in windows of 0.5 kb. (B) Protospacer hotspot peak resulting from a dsDNA break formed by the homing endonuclease I-SceI.(C) The overall number of protospacers around all Chi sites that are not included in the CRISPR or the Ter regions in a BL21-AIΔrecB strain. (D) The protospacer hotspot at the CRISPR region in the BL21-AIΔrecB strain is not confined by a Chi site (compare to the same hotspot in the WT strain, Fig. 3A). (E) Adaption levels in WT BL21-AI and BL21-AIΔrecB, ΔrecC or ΔrecD strains following overnight growth without arabinose induction of Cas1+2. (F) Percent new spacers derived from the self chromosome in the experiment described in Panel E. (G) Percent new spacers derived from the self chromosome in the presence of a plasmid that contains a cluster of 4 Chi sites (pChi) as compared to an identical plasmid that lacks Chi sites (pCtrl-Chi). For panels E-G, average and error margins for two biological replicates are shown.
Mentions: Several lines of evidence support this hypothesis. First, the orientation of the Chi sites at the protospacer peaks was always consistent with the dsDNA break occurring at the fork direction rather than the other side, and the first properly oriented Chi site upstream to the stalled fork was always the site of peak boundary (Fig. 3a-d). Second, even outside the strong protospacer hotspots, there was a significant asymmetry in protospacer density upstream and downstream Chi sites (Fig. 4a). The effect of this asymmetry was seen up to a distance of about 5-10kb from the Chi site, consistent with an average distance of ~5kb between Chi sites in the E. coli genome 22. Third, inducing a single, site specific dsDNA break in the chromosome using the homing endonuclease I-SceI resulted in a clear protospacer hotspot that peaked at the site of the dsDNA break and was confined by Chi sites in the proper orientations (Fig. 4b), directly linking dsDNA breaks to spacer acquisition hotspots. Fourth, co-immunoprecipitation assays suggested that Cas1 interacts with RecB and RecC 27 (although these interactions were not verified using purified proteins), supporting a model where the Cas1+2 complex is directly fed from RecBCD DNA degradation products. And finally, Cas1 was shown to efficiently bind ssDNA, which is amply generated during RecBCD DNA processing activity 23,27.

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