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. 2014 Feb;42(4):2577-90.
doi: 10.1093/nar/gkt1074. Epub 2013 Nov 22.

Phylogeny of Cas9 Determines Functional Exchangeability of dual-RNA and Cas9 Among Orthologous Type II CRISPR-Cas Systems

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Free PMC article

Phylogeny of Cas9 Determines Functional Exchangeability of dual-RNA and Cas9 Among Orthologous Type II CRISPR-Cas Systems

Ines Fonfara et al. Nucleic Acids Res. .
Free PMC article

Abstract

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

Figures

Figure 1.
Figure 1.
Phylogeny of representative Cas9 orthologs and schematic representation of selected bacterial type II CRISPR-Cas systems. (A) Phylogenetic tree of Cas9 reconstructed from selected, informative positions of representative Cas9 orthologs multiple sequence alignment is shown (Supplementary Figure S2 and Supplementary Table S2). The Cas9 orthologs of the subtypes classified as II-A, II-B and II-C are highlighted with shaded boxes. The colored branches group distinct proteins of closely related loci with similar locus architecture (15). Each protein is represented by the GenInfo (GI) identifier followed by the bacterial strain name. The bootstrap values are given for each node (see ‘Materials and Methods’ section). Note that the monophyletic clusters of subtypes II-A and II-B are supported by high bootstrap values. The scale bar for the branch length is given as the estimated number of amino acid substitution per site. (B) Genetic loci of type II (Nmeni/CASS4) CRISPR-Cas in Streptococcus pyogenes SF370, Streptococcus mutans UA159, Streptococcus thermophilus LMD-9 *(CRISPR3), **(CRISPR1), Campylobacter jejuni NCTC 11168, Neisseria meningitidis Z2491, Pasteurella multocida Pm70 and Francisella novicida U112. Red arrow, transcription direction of tracrRNA; blue arrows, cas genes; black rectangles, CRISPR repeats; green diamonds, spacers; thick black line, leader sequence; black arrow, putative pre-crRNA promoter; HP, Hypothetical Protein. The colored bars represented on the left correspond to Cas9 tree branches colors. The transcription direction and putative leader position of C. jejuni and N. meningitidis pre-crRNAs were derived from previously published RNA sequencing data (15). The CRISPR-Cas locus architecture of P. multocida was predicted based on its close similarity to that of N. meningitidis and further confirmed by bioinformatics prediction of tracrRNA based on a strongly predicted promoter and a transcriptional terminator as described in (15). Type II CRISPR-Cas loci can differ in the cas gene composition, mostly with cas9, cas1 and cas2 being the minimal set of genes (type II-C, blue), sometimes accompanied with a fourth gene csn2a/b (type II-A, yellow and orange) or cas4 (type II-B, green). The CRISPR array can be transcribed in the same (type II-A, yellow and orange) or in the opposite (types II-B and C, blue and green) direction of the cas operon. The location of tracrRNA and the direction of its transcription differ within the groups (compare type II-A of S. thermophilus** with type II-A from the other species indicated here (yellow) and compare type II-C of C. jejuni with type II-C of N. meningitidis and P. multocida (blue)).
Figure 2.
Figure 2.
RNase III is a general executioner of tracrRNA:pre-crRNA processing in type II CRISPR-Cas. Northern blot analysis of total RNA from S. pyogenes WT, Δrnc and Δrnc complemented with rnc orthologs or mutants (truncated rnc and inactivated (dead) (D51A) rnc) probed for tracrRNA (top) and crRNA repeat (bottom). RNA sizes in nucleotide and schematic representations of tracrRNA (red-black) and crRNA (green-black) are indicated on the right (16). The vertical black arrows indicate the processing sites. tracrRNA-171 nt and tracrRNA-89 nt forms correspond to primary tracrRNA transcripts. The presence of tracrRNA-75 nt and crRNA 39-42 nt forms indicates tracrRNA and pre-crRNA co-processing. S. pyogenes tracrRNA and pre-crRNA are coprocessed by all analyzed RNase III orthologs. The truncated version and catalytic inactive mutant of S. pyogenes RNase III are both deficient in tracrRNA:pre-crRNA processing.
Figure 3.
Figure 3.
Conserved motifs of Cas9 are required for DNA interference but not for dual-RNA processing by RNase III. (A) Schematic representation of S. pyogenes Cas9. The conserved HNH and splitted RuvC motifs and analyzed amino acids are indicated. (B) Northern blot analysis of total RNA from S. pyogenes WT, Δcas9 and Δcas9 complemented with pEC342 or pEC342 containing cas9 WT or mutant genes, probed for tracrRNA and crRNA repeat. Maturation of tracrRNA and pre-crRNA generating tracrRNA-75 nt and crRNA-39-42 nt forms is observed in all Δcas9 strains complemented with the cas9 mutants. (C) In vivo protospacer targeting. Transformation assays of S. pyogenes WT and Δcas9 with pEC85 (vector), pEC85Ωcas9 (cas9), pEC85ΩspeM (speM), and pEC85ΩtracrRNA-171 nt plasmids containing speM and cas9 mutants. The CFUs per µg of plasmid DNA were determined in at least three independent experiments. The results ±SD of technical triplicates of one representative experiment are shown. Cas9 N854A is the only mutant that did not tolerate the protospacer plasmid as observed for WT Cas9, indicating that this residue is not involved in DNA interference. (D) In vitro plasmid cleavage. Agarose gel electrophoresis of plasmid DNA (5 nM) containing speM protospacer (pEC287) incubated with 25 nM Cas9 WT or mutants in the presence of equimolar amounts of dual-RNA-speM (see ‘Materials and Methods’ section). Cas9 WT and N854A generated linear cleavage products while the other Cas9 mutants created only nicked products. M, 1 kb DNA ladder (Fermentas); oc: open circular, li: linear; sc: supercoiled.
Figure 4.
Figure 4.
Cas9 from closely related CRISPR-Cas systems can substitute the role of S. pyogenes Cas9 in RNA processing by RNase III. (A) Schematic representation of Cas9 from selected bacterial species. The protein sizes and distances between conserved motifs (RuvC and HNH) are drawn in scale. See Supplementary Figure S2. (B) Northern blot analysis of total RNA extracted from S. pyogenes WT, Δcas9 and Δcas9 complemented with pEC342 (backbone vector containing tracrRNA-171 nt and the cas operon promoter from S. pyogenes) or pEC342-based plasmids containing cas9 orthologous genes, probed for tracrRNA and crRNA repeat. Mature forms of S. pyogenes tracrRNA and pre-crRNA are observed only in the presence of S. pyogenes Cas9 WT or closely related Cas9 orthologs from S. mutans and S. thermophilus*.
Figure 5.
Figure 5.
Cas9 orthologs cleave DNA in the presence of their cognate dual-RNA and specific PAM in vitro. (A) Logo plot of protospacer adjacent sequences derived from BLAST analysis of spacer sequences for selected bacterial species. The logo plot gives graphical representation of most abundant nucleotides downstream of the protospacer sequence. The numbers in brackets correspond to the number of analyzed protospacers. (B) DNA substrates designed for specific PAM verification. Based on the logo plot for each species, plasmid DNA substrates were designed to contain the speM protospacer and the indicated sequence downstream, either comprising (PAM+) or not (PAM−) the proposed PAM. The predicted PAMs were verified by cleavage assays narrowing down the necessary nucleotides for activity (data not shown); therefore the sequence used differs slightly from the logoplot shown in (A). The high abundance of other nucleotides not being part of the PAM can be explained by redundancy of the coding sequences containing the protospacers, and by the limited number of found protospacer targets. The last column shows the PAM sequence for each species, which was already published (no symbol) or derived from this work (#). (C) In vitro plasmid cleavage assays by dual-RNA:Cas9 orthologs on plasmid DNA with the 10-bp protospacer adjacent sequence (summarized in (B)). Each Cas9 ortholog in complex with its cognate dual-RNA cleaves plasmids containing the corresponding species-specific PAM (PAM+). No cleavage is observed with plasmids that did not contain the specific PAM (PAM−). li: linear cleavage product, sc: supercoiled plasmid DNA.
Figure 6.
Figure 6.
Cas9 and dual-RNA coevolved. (A) In vitro plasmid cleavage assays using S. pyogenes Cas9 in complex with orthologous dual-RNA (upper panel) and orthologous Cas9 enzymes in complex with S. pyogenes dual-RNA (lower panel). Plasmid DNA containing protospacer speM and S. pyogenes PAM (NGG) was incubated with different dual-RNAs in complex with S. pyogenes Cas9. tracrRNA and crRNA-repeat sequences of the dual-RNAs are from the indicated bacterial species, with crRNA spacer targeting speM. In the lower panel, plasmid DNA containing speM protospacer and the specific PAM was incubated with Cas9 orthologs in complex with S. pyogenes dual-RNA. S. pyogenes Cas9 can cleave plasmid DNA only in the presence of dual-RNA from S. pyogenes, S. mutans and S. thermophilus* (yellow). Dual-RNA from S. pyogenes can mediate DNA cleavage only with Cas9 from S. pyogenes, S. mutans and S. thermophilus* (yellow). li: linear cleavage product; sc: supercoiled plasmid DNA. (B) Summary of Cas9 and dual-RNA orthologs exchangeability. Specific PAM sequences were used according to Figure 5. The color code reflects the type II CRISPR-Cas subgroups (Figure 1). +++: 100–75% cleavage activity; ++: 75–50% cleavage activity; +: 50–25% cleavage activity; −: 25–0% cleavage activity observed under the conditions tested. Cas9 and dual-RNA duplexes from the same type II group can be interchanged and still mediate plasmid cleavage providing that the PAM sequence is specific for Cas9. See also Supplementary Figure S10.

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