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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

Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacersDNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1+2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to WT arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location, and source of newly acquired spacers.
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Figure 6: Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacersDNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1+2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to WT arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location, and source of newly acquired spacers.

Mentions: We set out to understand the mechanism governing the self/non-self discrimination of the DNA source for spacer acquisition during the adaptation stage. For this, we used a previously described experimental system that monitors spacer acquisition in vivo in the E. coli type I-E CRISPR system 7,12. In this system, cas1 and cas2 are carried on a plasmid (pCas1+2) and their expression is regulated by an arabinose-inducible T7 RNA polymerase (Extended Data Fig. 1). We have previously shown that expression of Cas1+2 in this system leads to spacer acquisition, i.e., expansion of the chromosomally encoded CRISPR I array in E. coli BL21-AI 7. Since this strain of E. coli harbors a CRISPR array but lacks any cas genes on its genome, this system is interference-free, and thus does not allow ‘primed’ CRISPR adaptation 9,10,14,15.


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)

Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacersDNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1+2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to WT arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location, and source of newly acquired spacers.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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

Figure 6: Graphic overview of the procedure for characterizing the frequency and sequence of newly acquired spacersDNA from cultures of either E. coli K-12 (left) or E. coli BL21-AI (right) strains expressing Cas1+2 from two different plasmids were used as templates for PCR. Round 1 was used to determine the frequency of spacer acquisition by comparing occurrences of expanded arrays to WT arrays. Round 2 amplified only the expanded arrays and, followed by deep sequencing, was used to determine the sequence, location, and source of newly acquired spacers.
Mentions: We set out to understand the mechanism governing the self/non-self discrimination of the DNA source for spacer acquisition during the adaptation stage. For this, we used a previously described experimental system that monitors spacer acquisition in vivo in the E. coli type I-E CRISPR system 7,12. In this system, cas1 and cas2 are carried on a plasmid (pCas1+2) and their expression is regulated by an arabinose-inducible T7 RNA polymerase (Extended Data Fig. 1). We have previously shown that expression of Cas1+2 in this system leads to spacer acquisition, i.e., expansion of the chromosomally encoded CRISPR I array in E. coli BL21-AI 7. Since this strain of E. coli harbors a CRISPR array but lacks any cas genes on its genome, this system is interference-free, and thus does not allow ‘primed’ CRISPR adaptation 9,10,14,15.

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