Essential requirements for the detection and degradation of invaders by theHaloferax volcaniiCRISPR/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.

The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium

“…Interestingly, they found that plasmid copy number has no effect on the plasmid transformation rate, but the origin of replication (ori) used has an effect. Plasmids with the pHV1 ori were degraded by the CRISPR-Cas system (few transformants), while plasmids with the pHV2 ori were successfully transformed into cells (64). This observation is analogous to the results obtained with the nonrandom MOB-family conjugative plasmid spacer acquisition in CRISPRs (12).…”

“…In another study, Maier and coworkers tested the influence of plasmid copy number and the origin of replication on CRISPR-Cas type I-B interference in H. volcanii (64). Interestingly, they found that plasmid copy number has no effect on the plasmid transformation rate, but the origin of replication (ori) used has an effect.…”

“…Moreover, these plasmid-invader tests allowed the determination of possible protospacer mutations required to escape CRISPR-Cas systems or the identification of PAM sequences required for CRISPR adaptation/interference steps. These kinds of experiments have been used successfully with many different organisms such as E. coli (25,47,(60)(61)(62)(63), Haloarcula hispanica (48), Haloferax volcanii (56,57,64), P. atrosepticum (49), Sulfolobus spp. (58,65), Staphylococcus epidermidis (14), Streptococcus agalactiae (66), S. pyogenes (67), S. thermophilus (30), and Thermococcus kodakarensis (68).…”

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

“…CRISPR RNAs (crRNAs) are produced by processing of CRISPR locus transcripts that arise from promoter elements in the leader region (Jansen et al 2002;Tang et al 2002). Each crRNA contains a guide region comprised of invader-derived sequences that allow crRNA-Cas protein effector complexes to recognize and destroy invader nucleic acids (Barrangou et al 2007;Marraffini and Sontheimer 2008;Hale et al 2009Hale et al , 2012Garneau et al 2010;Jore et al 2011;Lintner et al 2011b;Wiedenheft et al 2011a;Fischer et al 2012;Gasiunas et al 2012;Jinek et al 2012;Westra et al 2012a;Elmore et al 2013;Maier et al 2013;Mulepati and Bailey 2013;Peng et al 2013Peng et al , 2015Sinkunas et al 2013;Plagens et al 2014).…”

Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

“…22 For the type I-B system of the archaeon Haloferax volcanii matching at positions 1-5, 7-10 and 13 was shown to be critical for effective interference. 23 However, more recent studies on the latter type I-B, I-E and I-F systems have suggested that less perfect annealing may still be effective, depending on the sequence of the protospacer and on the spacer-specific crRNA yields. [22][23][24][25] In contrast, studies on crRNA-protospacer annealing in Sulfolobus species have indicated that a much lower level of base pair matching can still produce effective interference.…”