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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

The protein product of T7 gene 5.9 inhibits spacer acquisition activityE. coli BL21-AI strains harboring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% L-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.
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Figure 10: The protein product of T7 gene 5.9 inhibits spacer acquisition activityE. coli BL21-AI strains harboring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% L-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.

Mentions: To test whether spacer acquisition indeed depends on the activity of the RecBCD complex, we used E. coli strains in which recB, recC or recD were deleted. Deep-sequencing-based quantification of spacer acquisition rates in these mutants showed reduced acquisition in all of these deletion strains (Fig. 4e; Extended Data Table 3a). Moreover, analysis of chromosomal protospacers in these mutants showed loss of spacer acquisition asymmetry near Chi sites (Fig. 4c), resulting in broader protospacer hotspots on the self chromosome (Fig. 4d). In accordance, the fraction of spacers derived from the self chromosome was ~10-fold higher in the recB, recC and recD deletion strains as compared to the WT strain (Fig. 4f; Extended Data Table 3a). These results show that CRISPR adaptation is partially dependent on the activity of the RecBCD dsDNA break repair complex, and that this activity is also responsible for some of the self/non-self discrimination properties of the CRISPR adaptation process. Consistent with these results, expression of a RecBCD inhibitor protein, the product of gene 5.9 of the T7 bacteriophage 28, showed reduced spacer acquisition as compared to exogenous expression of a control protein (Extended Data Fig. 5).


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)

The protein product of T7 gene 5.9 inhibits spacer acquisition activityE. coli BL21-AI strains harboring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% L-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 10: The protein product of T7 gene 5.9 inhibits spacer acquisition activityE. coli BL21-AI strains harboring pBAD-Cas1+2 and pBAD33-gp5.9 (lane 1) or pBAD33 vector control (lane 2) were grown overnight in the presence of inducers (0.4% L-arabinose). Gel shows PCR products amplified from the indicated cultures using primers annealing to the leader and to the fifth spacer of the CRISPR array. Results represent one of three independent experiments.
Mentions: To test whether spacer acquisition indeed depends on the activity of the RecBCD complex, we used E. coli strains in which recB, recC or recD were deleted. Deep-sequencing-based quantification of spacer acquisition rates in these mutants showed reduced acquisition in all of these deletion strains (Fig. 4e; Extended Data Table 3a). Moreover, analysis of chromosomal protospacers in these mutants showed loss of spacer acquisition asymmetry near Chi sites (Fig. 4c), resulting in broader protospacer hotspots on the self chromosome (Fig. 4d). In accordance, the fraction of spacers derived from the self chromosome was ~10-fold higher in the recB, recC and recD deletion strains as compared to the WT strain (Fig. 4f; Extended Data Table 3a). These results show that CRISPR adaptation is partially dependent on the activity of the RecBCD dsDNA break repair complex, and that this activity is also responsible for some of the self/non-self discrimination properties of the CRISPR adaptation process. Consistent with these results, expression of a RecBCD inhibitor protein, the product of gene 5.9 of the T7 bacteriophage 28, showed reduced spacer acquisition as compared to exogenous expression of a control protein (Extended Data Fig. 5).

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