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, 10 (5), 726-37

The tracrRNA and Cas9 Families of Type II CRISPR-Cas Immunity Systems

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The tracrRNA and Cas9 Families of Type II CRISPR-Cas Immunity Systems

Krzysztof Chylinski et al. RNA Biol.

Abstract

CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.

Keywords: CRISPR-Cas; Cas9 (Csn1); RNA maturation; RNA processing; adaptive immunity; bacteria; mobile genetic elements; small non-coding RNA; tracrRNA; type II system.

Figures

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Figure 1. Phylogenetic tree of representative Cas9 sequences. See also Fig. S1 and Table S1. Bootstrap values calculated for each node are indicated. Same color branches represent selected subclusters of similar Cas9 orthologs. CRISPR repeat length in nucleotides, average Cas9 protein size in amino acids (aa) and consensus locus architecture are shown for every subcluster. *- gi|116628213, **- gi|116627542, †- gi|34557790, ‡- gi|34557932. Type II-A is characterized by cas9-csx12, cas1, cas2, cas4. Type II-B is characterized by cas9, cas1, cas2 followed by a csn2 variant. Type II-C is characterized by a conserved cas9, cas1, cas2 operon.
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Figure 2. Architecture of type II CRISPR-Cas from selected bacterial species. The vertical color bars group the loci that code for Cas9 orthologs belonging to the same tree subcluster (compare with Figure 1). Thick black bar, leader sequence; black rectangles and green diamonds, repeat-spacer array. Note, that for simplicity the depicted repeat-spacer arrays do not represent the actual amount of spacers. Predicted anti-repeats are represented by arrows indicating the direction of putative tracrRNA ortholog transcription; red and yellow, high and low complementarity to the pre-crRNA repeat, respectively. Note that for the loci that were not verified experimentally, the CRISPR repeat-spacer array is considered here to be transcribed from the same strand as the cas operon. The transcription direction of the putative tracrRNA ortholog is indicated accordingly. *- gi|116628213, **- gi|116627542, †- gi|34557790, ‡- gi|34557932.
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Figure 3. tracrRNA and pre-crRNA co-processing in selected type II CRISPR-Cas systems. CRISPR loci architectures with verified positions and directions of tracrRNA and pre-crRNA transcription are shown. Black sequences, pre-crRNA repeats; red sequences, tracrRNA sequences base-pairing with crRNA repeats. Putative RNA processing sites as revealed by RNA sequencing are indicated with arrowheads. For each locus, arrowhead sizes represent relative amounts of the retrieved 5′ and 3′-ends (see Table S3). Blue arrowhead pairs represent putative RNase III co-processing sites with dark blue indicating the putative primary processing sites.
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Figure 4. Sequence diversity of tracrRNA orthologs. tracrRNA sequence multiple alignment. S. thermophilus and S. thermophilus2, tracrRNA associated with gi|116628213 and gi|116627542 Cas9 orthologs, accordingly. Black, highly conserved; dark gray, conserved; light gray, weakly conserved. Predicted consensus structure is depicted on the top of the alignment. Arrows indicate the nucleotide co-variations. S. pyogenes SF370, S. mutans UA159, L. innocua Clip11262, C. jejuni NCTC 11168, F. novicida U112 and N. meningitidis Z2491 tracrRNAs were validated by RNA sequencing and northern blot analysis.S. thermophilus LMD-9 tracrRNA was validated by northern blot analysis. P. multocida Pm70 tracrRNA was predicted from high similarity of the CRISPR-Cas locus with that of N. meningitidis Z2491. M. mobile 163K tracrRNA was predicted in silico from strong predictions of transcriptional promoter and terminator.

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