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CRISPR-based adaptive and heritable immunity in prokaryotes

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1 CRISPR-based adaptive and heritable immunity in prokaryotes
John van der Oost, Matthijs M. Jore, Edze R. Westra, Magnus Lundgren, Stan J.J. Brouns  Trends in Biochemical Sciences  Volume 34, Issue 8, Pages (August 2009) DOI: /j.tibs Copyright © 2009 Elsevier Ltd Terms and Conditions

2 Figure 1 The CRISPR–Cas system. The figure shows the make-up of a CRISPR–Cas locus in a prokaryote chromosome. (a) cas (CRISPR-associated) genes (block arrows; color scheme defined in Figure 2) are located adjacent to a CRISPR, which consists of a leader sequence (L), followed by a variable number of short repeats (R; e.g. 29 nt in E. coli K12) separated by variable spacer sequences (S1–Sn; e.g. 32 nt in E. coli K12). The spacer sequences often match those of fragments from virus genomes and plasmids, suggesting that they are often derived from these sources [26]. (b) Infection by mobile genetic elements occasionally leads to integration in the chromosomal CRISPR of a new spacer (S0) that is derived from the invading genome, providing the host with acquired resistance against the corresponding invader [19]. The corresponding sequence of the mobile genomic element is referred to as a proto-spacer (PS); a proto-spacer adjacent motif (PAM) has also frequently been demonstrated [36–39]. Trends in Biochemical Sciences  , DOI: ( /j.tibs ) Copyright © 2009 Elsevier Ltd Terms and Conditions

3 Figure 2 Comparative analysis of cas genes. In this figure, the core cas genes are indicated by single numbers: cas1 (1), cas2 (2), cas3 (3; 3N, nuclease domain; 3H, helicase domain), cas4 (4), cas5e, -t, -a and -h (5e, -t, -a and -h, in which the letter refers to CRISPR subtype) and cas6 (6). Similar colors indicate the proposed related function for the gene product: yellow, CRISPR adaptation (i.e. Cas1, Cas2, Cas4); blue, binding and processing of pre-crRNA to crRNA (i.e. the Cascade-like complex and RAMPs; the hatching patterns indicate sequence similarity [22,23]); purple, role in interference (i.e. Cas3-like); gray, putative novel polymerase (i.e. COG1335); white, no predicted function; dashed arrows indicate genes that have not been classified as cas. (a) The classification of CRISPR subtypes (listed to the left of each example species) is on the basis of the clustering of different cas genes, as originally proposed by Haft et al. [22] and adopted by the Integrated Microbial Genomes data management system ( Generally in good agreement with this grouping, independent analyses have been performed by Makarova et al. [23] based on Cas1 phylogeny (proposing at least seven Cas systems, termed CASS 1 to CASS 7) and by Kunin et al. [25] based on CRISPR repeat sequences (12 major clusters). Genes are drawn as block arrows (not to scale); bold arrows indicate that there are published structural and/or functional analyses of the Cas proteins that belong to a particular subtype. The products of the genes encoding the Cascade complex (see Figure 3) of subtype-E are indicated (CasA to CasE). Other abbreviations [22]: Cse (E. coli subtype, E. coli K12 operon; CASS-2), Csy (Yersinia pestis subtype, Pseudomonas aeruginosa PA14. operon; CASS-3), Csd (Desulfovibrio vulgaris subtype and operon; CASS-1), Csm (Mycobacterium tuberculosis subtype, Staphylococcus epidermidis operon; resembles CASS-6), Cst (Thermotoga neapolitana subtype, Pyrococcus furiosus operon; resembles CASS-7), Csn (Neisseria meningitidis subtype, Streptococcus thermophilus operon; CASS-4), Csa (Aeropyrum pernix subtype, Archaeoglobus fulgidus operon; CASS-5), Csh (Haloarcula marismortui subtype and operon; resembles CASS-7); Cmr (RAMP module consisting of RAMP proteins and the above-mentioned novel polymerase). (b) Examples of fusion and fission of cas genes. (i) Shows a typical cas1 gene, (ii) the fusion of the cas1 gene with a gene for an RT (retron-type reverse transcriptase [COG3344] [23]; e.g. as occurs in Chlorobium phaeobacteroides DSM266) and (iii) the fusion of the cas1 gene with a cas4 gene (e.g. as occurs in Geobacter sulfurreducens; the fusion clusters with cas2, cas3 and csx genes). (iv) Shows the typical cas3 gene encoding a nuclease domain (N) and a helicase domain (H), (v) the fission and rearrangement of the cas3N and H fragments (as found in CRISPR subtype-A) and (vi) the fusion of the cas3 gene with cas2 (as found in CRISPR subtype-Y) [23]. Trends in Biochemical Sciences  , DOI: ( /j.tibs ) Copyright © 2009 Elsevier Ltd Terms and Conditions

4 Figure 3 A working model for the CRISPR–Cas defense system. Three distinct functional stages are recognized. (1) CRISPR adaptation: alien DNA is recognized (red), processed and integrated as new spacer into the chromosomal CRISPR locus (yellow genes (block arrows) encode Cas proteins proposed to be involved). (2) CRISPR expression: transcription of CRISPR, binding of pre-crRNA to Cascade complex (blue) and processing to crRNAs. (3) CRISPR interference: Cas3 (purple) and Cascade loaded with crRNA (blue and red) associate and/or degrade the target nucleic acid (DNA). Trends in Biochemical Sciences  , DOI: ( /j.tibs ) Copyright © 2009 Elsevier Ltd Terms and Conditions

5 Figure 4 Generation of crRNAs. (a) Endonuclease cleavage (indicated by black arrow head) of the CRISPR repeat in E. coli K12 (by the CasE [Cse3] subunit of the Cascade complex [CasABCDE]), in S. epidermidis (possibly by Cas6) and in P. furiosus (by Cas6). Inverted repeat sequences suggested to be involved in secondary structure formation are underlined [25]. To some extent, the analysis of Sulfolobus repeats [39] suggests that they also fit into this scheme (not shown). (b) Ribbon diagram with a ‘rainbow’ coloration of the 3D structures of the CRISPR RNA endonucleases Cse3 (CasE) from T. thermophilus [33] and Cas6 from P. furiosus [34]. The duplicated ferredoxin fold (a common α+β protein fold with a typical βαββαβ secondary structure) and the glycine-rich loop (red) near the C-terminus typify this protein family. Divalent metal-ion independent phosphodiester bond cleavage is carried out by the catalytic histidine residue (H26) in Cse3 and the catalytic triad consisting of tyrosine (Y31), histidine (H46) and lysine (K52) of Cas6. The structures are readily superimposed with a backbone RMSD of 0.89Å. Trends in Biochemical Sciences  , DOI: ( /j.tibs ) Copyright © 2009 Elsevier Ltd Terms and Conditions


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