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Thus, the size distribution of the newly acquired spacers closely matched that of the naturally acquired spacers both in WT and OE strains, and 37 bp spacers were predominately acquired in all seven CRISPR loci. To search for PAM sequences involved in adaptation, we constructed sequence logos 33 of the flanking sequences upstream and downstream of uniquely mapped protospacers.

The bioinformatically-predicted PAM for the seven P. During DNA interference, wild type P. Analysis of PAM sequences in adaptation. A Newly-acquired spacers in each CRISPR array were used to search the genome and plasmids in order to identify the corresponding protospacers, and upstream and downstream sequences were extracted and used to generate consensus motifs on both strands of DNA. Four bp of flanking sequence on each side of the protospacers is shown. C Based on the observed consensus sequences, a model of the ideal upstream and downstream motifs was made.

In contrast, when using pJFW18, only 0. Both plasmids are a similar size pYS3-Pgdh is bp, pJFW18 is bp , and it has been reported that the copy number of both pJFW18 and pYS3 is 1—2 copies per chromosome 31 , 42 , so the amount of plasmid DNA is expected to be similar in all experiments.

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These results indicate that spacers are preferentially acquired from the rolling circle replication plasmid compared to the theta replication plasmid. Spacers are preferentially acquired from rolling circle replication plasmids. Both theta A and rolling circle B replication plasmids were used in these studies.

Cartoon models shown here illustrate how replication is thought to occur in each plasmid type. A Theta plasmid replication initiates synthesis at a replication origin OriC. Replication extends the leading strand continuously while synthesizing the lagging strand discontinuously. B Rolling circle replication is initiated by a Rep protein, which nicks the double-strand origin Replication proceeds using the un-nicked strand as a template displacing the nicked strand.

C Spacers from expanded arrays were aligned to both the P. D Alignment of protospacers to the plasmid shows an unequal distribution. Green bars show origin of DNA replication. Protospacers on the plus and minus strand are indicated in blue and pink, respectively. We next examined spacer distribution along the plasmids. Together these results suggest that certain steps in DNA replication that are unique to the rolling circle mechanism make this type of plasmid a good target for spacer acquisition.

As described in more depth below, significant protospacer hotspots in the P. However, the other peaks could not be explained by DNA content.

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Aligned protospacers are not evenly distributed across the P. A and B Distribution of protospacers on the P. Protospacers on the plus and minus strand are indicated in blue and pink respectively. Black bars show regions where protospacers are significantly enriched over the background, as detected by the peak-calling software from the HOMER package.

A Distribution of protospacers across the full length of the chromosome.

B and C Protospacers are enriched around areas where homology exists between the chromosome and the plasmid. Prominent clusters of protospacers are observed extending s of bps out from the region of homology. D Model of homologous recombination occurring at the homologous region Pgdh between plasmids and the P. Recombination results in integration of the plasmid into the chromosome.

E PCR results confirm that homologous recombination between the plasmid and chromosome occurs. Top panel shows amplified products of plasmid-integrated chromosome. Middle panel shows internal control of chromosome and bottom panel shows internal control of the plasmid. Because the Gdh promoter bp exists on both the P.

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To test that, we conducted a follow-up experiment using the pYS3-Pslp plasmid, which is identical to pYS3-Pgdh except that the Gdh promoter is replaced by the Slp promoter. The directionality of the hotspot changes, likely reflecting that the Slp promoter was cloned into the plasmid in the opposite orientation with respect to the origin of replication. We verified that homologous recombination does occur between the plasmid and chromosome at the point of the Gdh promoter, by PCR using a set of primers in which the forward primer anneals within Rep75 of the pYS3 plasmid and the reverse primer within the ORF of the Gdh gene on the chromosome.

This homologous recombination also occurs between the pJFW18 plasmid and the chromosome, but with significantly reduced recombination efficiency Supplementary Figure S5. Taken together, the results indicate that DNA nicking and rolling circle replication of the plasmid following homologous recombination into the host chromosome induced enhanced protospacer generation of adjacent host genome self-targeted sequences.

The P. In all, 24 of the 29 active transposons had associated protospacer peaks in at least one experiment, while only three of the 16 inactive transposons had such a peak. This strongly suggests that some aspect of transposon activity promotes protospacer generation. A co-integrated product is formed between the donor IS element and the target DNA and is subsequently resolved by recombination to generate a new transposon at a new site while preserving the original transposon copy Protospacers are enriched for transposons.

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A Cartoon model of the mechanism for transposon replication. Target DNA is also nicked and the transposon joins to the target.

The transposon intermediate is resolved by recombination. The original and target sites are separated, each harboring one copy of the transposon. B and C Protospacers are significantly enriched around active transposons. Black bars show areas were protospacers are significantly enriched, as detected by peak finding software in the HOMER package. Transposons that were previously described as active 44 were 10 times more likely to have an associated peak than those described as inactive red. B and C Protospacers on the plus and minus strand are indicated in blue and pink respectively.

Black bars show areas were protospacers are significantly enriched, as detected by peaking finding software in the HOMER package. Moreover, R-loop formation can lead to nicking of the displaced DNA strand as well as double-strand breaks when replication forks stall at R-loop structures Our results reinforce the idea that DNA in an R-loop also provides a potential source of protospacers.

By this standard, no matches were found. It is understood that this acquisition would likely be guided such that the new CRISPR spacers are suitable for later targeting of invaders.

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However, the steps underlying adaptation are not well studied, and evidence and mechanisms for such guidance is lacking. Here we report the first evidence that P. The uptake of new spacers was dependent both on the presence and expression level of the presumed set of adaptation proteins: Cas1, Cas2 and Cas4.

Instead, observations of spacer distribution along the genome and plasmids suggests that nicked or broken DNA may provide the best source of protospacers, irrespective of how the free DNA ends are generated. Interestingly, we found that highly sampled protospacers from either invading plasmid or the host genome are derived from specific regions of DNA expected or known to experience DNA nicking or double-strand breaks.

In particular, site-specific nicking events that apparently contribute to protospacer generation in P. Moreover, the observed protospacer hotspot region that overlaps the massively transcribed single rDNA loci of P. In the E. These hotspots are RecBCD-dependent and caused by double-strand breaks at presumed stalled replication forks.

In the P. Prokaryotes must cope with both intercellular mobile genetic elements such as viruses and plasmids as well as intracellular mobile genetic elements including transposons that each can harm or kill cells or lead to genome instability. The apparent requirement for free DNA ends for efficient protospacer acquisition may have evolved to more effectively identify both classes of mobile genetic element. Unlike host chromosomes that tend to be circular i. Moreover, specific nicking reactions are often employed to enable transfer of DNA from one cell to the next e.

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This tendency for intracellular mobile genetic element DNA to become linear or nicked within their host cells may make them vulnerable to spacer uptake by CRISPR—Cas adaptation machinery that appears to require free DNA termini for access to protospacers. The acquisition of self-targeting spacers typically leads to cell death lethal autoimmunity in prokaryotes 16 , 56 , Our approach of studying spacer acquisition in a cultured population of cells provides us with a steady-state view i.

Using a similar strategy, we previously found that Type II-A Cas9-based CRISPR—Cas systems also normally sample both chromosomal and extrachromosomal DNA and that cellular counter-selection through self-targeting and lethal autoimmunity provides the basis for the apparent specificity observed for CRISPR arrays normally lacking significant self-targeting sequences In Sulfolobus , conjugative plasmids are known to pass between cells and integrate into the genome of the recipient Though these specific events have not been described in Pyrococcus , similar harmful plasmid integration may exist and the recombination inadvertently induced in our assay may mimic such a scenario.

Together, these findings raise the interesting possibility that self-targeting by CRISPR—Cas systems, which eliminates specific cells from a population, may contribute to increased genomic stability through copy number control of transposons and prevention of the uptake of autonomously replicating mobile genetic elements into host chromosomes. In this view, self-targeting spacers may serve a positive role at times by eliminating cells that are undergoing detrimental changes to their genomes. We envision that the nicked or broken DNA molecules serve as entry points for nucleases and helicases that promote formation of an intracellular pool of heterogenous linear DNA fragments.

Subsequently, the adaptation machinery catalyzes the polarized addition of processed spacers at the leader end of the CRISPR array.