Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B

Abstract

To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5' handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.

Keywords: CRISPR/Cas; Haloferax volcanii; PAM; archaea; crRNA; seed sequence.

Figure 1. (A)Repeat sequences of the three CRISPR loci in H. volcanii. The repeat sequences of the three different CRISPR RNAs differ by one nucleotide (at position 23). The sequence of the CRISPR locus C repeat is shown. Positions that are identical in the P1 and P2 locus repeats are indicated with a dot. The pre-crRNA is processed in the repeat region directly upstream of nucleotide 23 (marked by the arrow). (B) Structure of the crRNA in Haloferax. The product of the pre-crRNA processing reaction is shown. It contains the spacer sequence and an eight-nucleotide 5′ handle, which originates from the upstream repeat. Because position 23 varies between the three CRISPR repeats, the first nucleotide of the 5′ handle is either a G (locus C), an A (locus P1) or a U (locus P2). Downstream of the spacer are the remaining 22 nucleotides of the downstream repeat, which can fold into a minimal stem loop.

Figure 2. A small stem loop structural motif is conserved across 22 haloarchaeal species. (A) Part of the predicted structure for the repeat from locus C is conserved throughout the haloarchaeal species (highlighted in yellow). The red line corresponds to the cleavage site just upstream of the 5′ crRNA tag. The G nucleotide that is cyan in color corresponds to the 23rd nucleotide, which is different in the P1 (A) and P2 (U) loci. (B and C) Multiple sequence alignments generated by LocARNA; the red columns correspond to conserved base pairs and the mustard yellow columns correspond to the presence of a compensatory base pair that conserves the consensus structure. The conserved structural motif from (A) is surrounded by the black box. (B) The larger group of haloarchaea with the conserved motif and a 4-nucleotide hairpin loop. (C) The smaller group with a 5-nucleotide hairpin loop. The conserved CG stem loop motif is surrounded by stabilizing base pairs in both groups.

Figure 3. Profile of reads across each CRISPR locus: crRNAs accumulate to varying degrees. The reads of the extracted crRNA are mapped to the three CRISPR RNA loci and the variation in their abundance is shown. The blue bars correspond to the spacer number. The number of reads (y-axis) is given on a log-scale. In locus P1, a large deletion of 23 spacers occurred in our lab strain, in comparison to the sequenced genome in the database. Due to the internal priming of the 3′ oligonucleotide used in the sequencing protocol, the 3′ ends generated do not represent the in vivo 3′ ends. For mapping, the reads that started with the 5′ repeat tag were extracted to a maximum length of 35 nucleotides.

Figure 4. A seed sequence is required to trigger an interference reaction. (A) To investigate the positions that are essential for successful interference, nucleotides were mutated or deleted (marked by a d) in the protospacer sequence. The PAM sequence is indicated in green. Variants that were recognized by the CRISPR/Cas machinery are marked with a “+,” variants that were not recognized as invader are labeled with a “–“. (B) The interaction between the crRNA and the invader plasmid is shown schematically. The crRNA and the plasmid form an R loop, in which the crRNA is able to base pair with the protospacer sequence. The PAM sequence is shown in green, the protospacer sequence in yellow. Base pairs that are essential for the interference reaction are shown in red, while non-essential base pairs are shown in grey.

Figure 5. Interference as a function of the invader plasmid origin. To analyze whether the efficiency of the interference reaction depends on the copy number, different types of vectors were used to clone the protospacer and PAM sequences. Haloferax cells were transformed with pTA409-PAM3-P1.1 (pyrE2 marker) and pTA232-PAM3-P1.1 (leuB marker) simultaneously. Plating on medium that was selective for both plasmids (ura- and leu-) resulted in very low transformation rates. Plating on medium selective only for plasmid pTA409-PAM3-P1.1 (ura-) resulted, likewise, in very low transformation rates, indicating that in both cases, the interference reaction was successful. In contrast, plating on medium that was selective only for plasmid pTA232-PAM3-P1.1 (leu-) resulted in full plates, indicating that the defense reaction was not active. Taken together, these data demonstrate that plasmid pTA409-PAM3-P1.1 is selectively degraded.