Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

Abstract

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9-crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.

Fig. 1.

The Cas9 protein copurifies with crRNA. (A) Schematic representation of the CRISPR3/Cas system of S. thermophilus DGCC7710. Four cas genes (cas9, cas1, cas2, csn2) are located upstream of the CRISPR repeat-spacer array, consisting of 13 repeat (R) sequences and 12 unique spacers (S1–S12). The tracrRNA, required for crRNA maturation in type II CRISPR/Cas systems (21), is located upstream of the cas9 gene and is encoded on the opposite DNA strand (shown by an arrow) with respect to the other elements of this system. (B) Schematic representation of heterologous loci in two plasmids used for the coexpression of the Cas9–crRNA complex. E. coli RR1 contained pCas9(−)SP1 (encoding Cas1, Cas2, Csn2, SP1, and tracrRNA) and pASKIBA-Cas9 (encoding the Strep-tagged version of Cas9) plasmids. (C) Northern blot analysis of Cas9–crRNA complexes using anti-crDNA oligonucleotide as a probe. M1, 84-nt oligodeoxynucleotide corresponding to the spacer1-repeat unit; M2, 42-nt synthetic oligoribonucleotide corresponding to the predicted S. thermophilus CRISPR3 crRNA (Fig. S2); crRNA (WT), crRNA isolated from the WT Cas9–crRNA complex; K1, crRNA (WT) treated with DNase I for 15 min; K2, crRNA (WT) treated with RNase I for 15 min; D31A, crRNA purified from the Cas9 D31A mutant complex; N891A, crRNA purified from the Cas9 N891A mutant complex.

Fig. 2.

The Cas9–crRNA complex cleaves dsDNA within the protospacer. (A) Oligoduplex substrate used in the cleavage assay. The 55-nt oligoduplex SP1 contains the protospacer1 (red letters), PAM (blue letters), and 10-nt flanking sequences on both sides identical to those in pSP1 plasmid. In the SP1 oligoduplex, the DNA strand complementary to the 5′-terminal fragment of crRNA (red letters) is termed the “(+)strand,” and the opposite DNA strand is termed the “(−)strand.” (B) Oligoduplex SP1 cleavage. The Cas9–crRNA complex (2.5 M) and 1 nM SP1 oligoduplex labeled with ³³P at the 5′ end of either the (+) or (–)strand were incubated in the reaction buffer [10 mM Tris⋅HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl₂, 0.1 mg/mL BSA] at 37 °C for various time intervals (30 s to 10 min), and reaction products were analyzed by 20% PAGE. Lanes M1 and M2 contain chemically synthesized 5′-end, ³³P-labeled, 37-nt and 18-nt oligodeoxynucleotides corresponding to the cleavage products of (−) and (+) DNA strands, respectively. Cleavage positions are indicated by arrows. (C) Schematic representation of pSP1 plasmid (16) used in the plasmid cleavage assay. (D) pSP1 plasmid cleavage. (Left) Agarose gel analysis of pSP1 cleavage products. FLL, full-length linear DNA cut at both strands; OC, open circular DNA nicked at one of the strands; SC, supercoiled plasmid DNA. Final reaction mixtures at 37 °C contained 2.5 nM of the pSP1 plasmid and 2.5 nM of the Cas9–crRNA complex in the reaction buffer (see section B). (Right) Direct-sequencing electropherograms of (+) (Upper) and (–) (Lower) strands of the pSP1 plasmid cleavage product.

Fig. 3.

A PAM is required for in vitro DNA binding and cleavage by the Cas9–crRNA complex. (A) Agarose gel analysis of plasmid DNA cleavage products. Three different plasmids—PAM+Protospacer+ (pSP1 containing both the protospacer and the PAM), PAM+Protospacer− (pUC18 containing multiple PAMs but no protospacer), and PAM-Protospacer+ [pSP1-pΔ (16) containing a protospacer without a PAM]—were incubated at 2.5-nM concentration with 2 nM of the Cas9–crRNA complex in the reaction buffer [10 mM Tris⋅HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl₂, 0.1 mg/mL BSA] at 37 °C for various time intervals, and reaction products were analyzed by agarose gel electrophoresis. FLL, full- length linear DNA cut at both strands; OC, open circular DNA nicked at one of DNA strands; SC, supercoiled plasmid DNA. (B) Time courses of (+)strand hydrolysis in the single-stranded and double-stranded oligodeoxynucleotides. Reactions containing 2 nM Cas9–crRNA and 1 nM of oligodeoxynucleotide were conducted at 37 °C in the reaction buffer (see section A). SP1 (filled circles) and SP1-pΔ (open squares) oligoduplexes were used as dsDNA. s(+)SP1 (open triangles) and s(+)SP1-pΔ (filled squares) were used as ssDNA. (C and D) dsDNA and ssDNA (+)strand) binding by the Cas9–crRNA complex. The reactions contained 0.5 nM of the ³³P-labeled ssDNA or dsDNA oligonucleotide and the protein at the concentrations indicated above each lane. After 15 min at room temperature, the samples were subjected to PAGE for 2 h and analyzed as described in Materials and Methods.

Fig. 4.

RuvC and HNH active-site motifs of Cas9 contribute to the cleavage of opposite DNA strands. (A) Localization of the conserved active-site motifs within Cas9 protein. Amino acid residues identified as crucial for Cas9 in vivo activity (16) are indicated. (B) Agarose gel analysis of pSP1 plasmid cleavage by Cas9 and mutant proteins. Reactions were performed as described in Materials and Methods. (C) Strand preference of the D31A mutant. Reactions were performed as described in Fig. 2A and Materials and Methods. The D31 mutant cleaves only the (+)strand of the SP1 oligoduplex. (D) Strand preference of the N891A mutant. The N891 mutant cleaves only the (−)strand of the SP1 oligoduplex. Cleavage positions are indicated by arrows.

Fig. 5.

Schematic arrangement and mechanism of crRNA-directed DNA cleavage by the Cas9–crRNA complex. Domain architecture of Cas9 is shown schematically at the top. The Cas9–crRNA complex binds to the dsDNA containing a PAM. crRNA binds to the complementary (+)strand, resulting in DNA strand separation and R-loop formation. In the ternary complex, the RuvC active site of Cas9 is positioned at the scissile phosphate on the unpaired (−)strand, and the HNH active site is located at the scissile phosphate on the DNA (+)strand bound to crRNA. Coordinated action of both active sites results in the double-strand break 3 nt upstream the PAM, generating blunt-ended DNA.

Fig. P1.

The Cas9–crRNA complex generates two distinct DNA nicks on opposing dsDNA strands that match the loaded small, interfering crRNA sequence. (A) After phage DNA entry into the cell, a piece of the invading DNA is inserted as a spacer into the CRISPR locus. (B) The CRISPR repeat-spacer array is transcribed and processed into short crRNAs. (C) The crRNA forms a ribonucleoprotein complex with Cas9, which recognizes invading DNA homologous to the crRNA sequence and mediates interference. (D) Invading DNA cleavage by Cas9–crRNA. In the presence of Mg²⁺ ions, the signature Cas9 protein nicks each DNA strand 3 nt upstream of the PAM sequence to generate blunt DNA ends through RuvC- and HNH-like active sites that act on separate DNA strands.