Materials and Methods

Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake inPyrococcus furiosus

MATERIALS AND METHODS P. furiosus strains and growth conditions Pyrococcus furiosus strain JFW02 (29) was used as wild type in this study. P. furiosus was grown anaerobically in a defined medium with cellobiose as the carbon source (30) at 90°C for 16–20 h in anaerobic culture bottles or on medium solidified with 1% (wt/vol) Gelrite (Research Products International) for 65 h. For growth of uracil auxotrophic strains, the defined medium contained 20 μM uracil. Plasmid transformation was performed as described previously (30). Pyrococcus furiosus strains were cultured anaerobically at 90°C to mid-to-late log phase in defined liquid medium. Pyrococcus furiosus culture was mixed with DNA at a concentration of 2.5 ng/μl culture and spread in 35 μl aliquots onto defined solid medium.Plasmid constructionPlasmids were constructed using standard cloning techniques. The sequences of oligonucleotides used in this study are shown in Supplemental Table S1. To generate the pYS3-Pslp plasmid, SimR of the pYS3 plasmid (31) was replaced by a P. furiosus slp-promoter (250 bp upstream of PF1399)-pyrF gene expression cassette. The insert for pMS32 plasmid was constructed by overlap PCR using JFW02 genomic DNA and pJE47 plasmid. The genome insertion cassette between PF1120 and CRISPR7, ∼0.7 kb flanking regions immediately upstream and downstream of the Cas2–Cas1–Cas4 gene cluster, with NotI-EcoRV sites was inserted into pHSG298. The Cas overexpression cassette, csg promoter–Cas2 (PF1117)—slp promoter–Cas4 (PF1119)—PRP synthetase promoter–Cas1 (PF1118), was cloned into pJE47. pHSG298 with the genome insertion cassette and the Cas overexpression cassette were digested by NotI and EcoRV and ligated to yield pMS32 plasmid. The plasmids were sequenced to confirm insert sequence and orientation.Strain constructionTo create a Cas1, Cas2, Cas4 protein overexpression strain (OE; TPF43), NruI-linearized pMS32 plasmid was transformed into the JFW02 (wt) strain. Two rounds of colony purification were performed by plating 10−3 dilutions of transformant cultures onto selective medium (without 20 μM uracil) and picking isolated colonies into selective liquid medium. Following marker replacement of the region of interest, 2.75 mM 5-FOA, a toxic PyrF substrate, was used to select for cells that had popped out the pyrF marker by homologous recombination between short regions of homology. The Cas1, Cas2 and Cas4 deletion strain (Δ strain; TPF55) was created using the pop-out marker replacement strategy as described previously (29). The transformed PCR products were generated by overlap PCR. The sequence of oligonucleotide used is showed in Supplemental Table S1.Spacer acquisition assay and high throughput sequencingTwenty colonies of P. furiosus strains transformed with plasmid were inoculated in 5 ml defined medium containing 20 μM uracil. Cultures were incubated at 90°C overnight. Genomic DNA was isolated from cells in 1 ml of overnight culture using quick-gDNA miniprep kit (Zymo Research). CRISPR arrays were amplified by PCR using a set of primers in which the forward primer annealed within the leader region of the CRISPR array and the reverse primer annealed to the existing spacer closest to the leader (referred to as the first round of PCR). If a new spacer was integrated into the CRISPR array, the resulting PCR product was longer due to the additional repeat and spacer sequence. These larger, expanded PCR products were separated from unexpanded products by gel electrophoresis followed by DNA recovery (Zymo Research). PCR primers included an overhang corresponding to part of the adapter necessary for Illumina sequencing. After size selection of the first PCR product, a second round of PCR was done to further amplify the expanded product. For very faint products, this second PCR was size selected and then a third round of PCR was done. Lastly, a nested PCR was done to add additional sequences corresponding to Illumina adapters and barcodes. Each experimental condition and replicate received a unique barcode (index) for multiplexing. The sequences of oligonucleotides used in the PCR reactions are listed in Supplemental Table S1.Final gel-purified amplicon libraries were ranked and pooled by PCR intensity, and the pooled DNA was purified and concentrated by ethanol precipitation. DNA pools were quantitated, normalized according to concentration and number of samples represented in the pool, and then combined to make a final pool for sequencing. Array libraries were sequenced on an Illumina MiSeq set to yield 200 × 100 paired-end reads; the 200 base read 1 sequences were used in this study.After sequencing, samples were de-multiplexed by index, and the sequence corresponding to a new (expanded) spacer was extracted from each read. To determine the source of these new spacers (i.e. to identify the protospacer sequence) we used Bowtie (32) to align the reads to a reference containing the genome and any plasmids used. For each experiment, the set of aligned protospacer sequences was then characterized with respect to length, GC content, and position on the genome or plasmid. We determined the proportion of new spacers derived from the genome versus a plasmid, and from the plus versus minus strand. Consensus sequences in the DNA upstream and downstream from the protospacer positions were identified by making sequence logos (33) from adjacent genomic sequences which were extracted using bedtools (34).Next, we created custom genome browser tracks (tools for this are available through the UCSC genome browser) to identify spatial patterns in the distribution of protospacers. Since the presence of a PAM sequence was shown to promote recruitment of a protospacer, we took into account the underlying distribution of PAM sequences along the genome and plasmids. In silico, we created a ‘PAMscape’ track in which a potential 37 bp protospacer is found immediately 3′ to all PAM sequences in the genome. Any clusters or ‘hotspots’ of protospacers that deviated from the background of PAM distribution were assumed to be due to mechanisms other than local sequence motifs. In addition to visually examining the tracks, we used the findPeaks software in the HOMER analysis package (35) to identify statistically significant clusters of protospacers. For this analysis we used a read-count normalized PAMscape as an input control and used the following analysis options to optimize the process for detecting variably-sized spacer peaks: -style histone, -size 500, -strand separate, -fragLength 40, -region.

Article TitleRole of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake inPyrococcus furiosus


To acquire CRISPR–Cas immunity against invasive mobile genetic elements, prokaryotes must first integrate fragments of foreign DNA into their genomic CRISPR arrays for use in future invader silencing. Here, we found that the hyperthermophilic archaeaon,Pyrococcus furiosus, actively incorporates DNA fragments (spacers) from both plasmid (foreign) and host genome (self) sequences into its seven CRISPR loci. The majority of new spacers were derived from DNA immediately downstream from a 5′-CCN-3′ protospacer adjacent motif (PAM) that is critical for invader targeting. Interestingly, spacers were preferentially acquired from genome or plasmid regions corresponding to active transposons, CRISPR loci, ribosomal RNA genes, rolling circle origins of replication, and areas where plasmids recombined with the host chromosome. A common feature of the highly sampled spacers is that they arise from DNA regions expected to undergo DNA nicking and/or double-strand breaks. Taken together with recent results from bacterial systems, our findings indicate that free DNA termini and PAMs are conserved features important for CRISPR spacer uptake in diverse prokaryotes and CRISPR–Cas systems. Moreover, lethal self-targeting by CRISPR systems may contribute to host genome stability by eliminating cells undergoing active transposon mobility or chromosomal uptake of autonomously replicating foreign mobile genetic elements.

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