Research Article

A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

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Science  17 Aug 2012:
Vol. 337, Issue 6096, pp. 816-821
DOI: 10.1126/science.1225829
  • Fig. 1

    Cas9 is a DNA endonuclease guided by two RNA molecules. (A) Cas9 was programmed with a 42-nucleotide crRNA-sp2 (crRNA containing a spacer 2 sequence) in the presence or absence of 75-nucleotide tracrRNA. The complex was added to circular or XhoI-linearized plasmid DNA bearing a sequence complementary to spacer 2 and a functional PAM. crRNA-sp1, specificity control; M, DNA marker; kbp, kilo–base pair. See fig. S3A. (B) Cas9 was programmed with crRNA-sp2 and tracrRNA (nucleotides 4 to 89). The complex was incubated with double- or single-stranded DNAs harboring a sequence complementary to spacer 2 and a functional PAM (4). The complementary or noncomplementary strands of the DNA were 5′-radiolabeled and annealed with a nonlabeled partner strand. nt, nucleotides. See fig. S3, B and C. (C) Sequencing analysis of cleavage products from Fig. 1A. Termination of primer extension in the sequencing reaction indicates the position of the cleavage site. The 3′ terminal A overhang (asterisks) is an artifact of the sequencing reaction. See fig. S5, A and C. (D) The cleavage products from Fig. 1B were analyzed alongside 5′ end-labeled size markers derived from the complementary and noncomplementary strands of the target DNA duplex. M, marker; P, cleavage product. See fig. S5, B and C. (E) Schematic representation of tracrRNA, crRNA-sp2, and protospacer 2 DNA sequences. Regions of crRNA complementarity to tracrRNA (orange) and the protospacer DNA (yellow) are represented. The PAM sequence is shown in gray; cleavage sites mapped in (C) and (D) are represented by blue arrows (C), a red arrow [(D), complementary strand], and a red line [(D), noncomplementary strand].

  • Fig. 2

    Cas9 uses two nuclease domains to cleave the two strands in the target DNA. (A) (Top) Schematic representation of Cas9 domain structure showing the positions of domain mutations. D10A, Asp10→Ala10; H840A; His840→Ala840. (Bottom) Complexes of WT or nuclease mutant Cas9 proteins with tracrRNA:crRNA-sp2 were assayed for endonuclease activity as in Fig. 1A. (B) Complexes of WT Cas9 or nuclease domain mutants with tracrRNA and crRNA-sp2 were tested for activity as in Fig. 1B.

  • Fig. 3

    Cas9-catalyzed cleavage of target DNA requires an activating domain in tracrRNA and is governed by a seed sequence in the crRNA. (A) Cas9-tracrRNA:crRNA complexes were reconstituted using 42-nucleotide crRNA-sp2 and truncated tracrRNA constructs and were assayed for cleavage activity as in Fig. 1B. (B) Cas9 programmed with full-length tracrRNA and crRNA-sp2 truncations was assayed for activity as in (A). (C) Minimal regions of tracrRNA and crRNA capable of guiding Cas9-mediated DNA cleavage (blue shaded region). (D) Plasmids containing WT or mutant protospacer 2 sequences with indicated point mutations (right) were cleaved in vitro by programmed Cas9 as in Fig. 1A (top-left) and used for transformation assays of WT or pre-crRNA–deficient S. pyogenes (bottom-left). The transformation efficiency was calculated as colony-forming units (CFU) per microgram of plasmid DNA. Error bars represent SDs for three biological replicates. (E) Plasmids containing WT and mutant protospacer 2 inserts with varying extent of crRNA-target DNA mismatches (right) were cleaved in vitro by programmed Cas9 (left). The cleavage reactions were further digested with XmnI. The 1880- and 800-bp fragments are Cas9-generated cleavage products. M, DNA marker.

  • Fig. 4

    A PAM is required to license target DNA cleavage by the Cas9-tracrRNA:crRNA complex. (A) Dual RNA-programmed Cas9 was tested for activity as in Fig. 1B. WT and mutant PAM sequences in target DNAs are indicated (right). (B) Protospacer 4 target DNA duplexes (labeled at both 5′ ends) containing WT and mutant PAM motifs were incubated with Cas9 programmed with tracrRNA:crRNA-sp4 (nucleotides 23 to 89). At the indicated time points (in minutes), aliquots of the cleavage reaction were taken and analyzed as in Fig. 1B. (C) Electrophoretic mobility shift assays were performed using RNA-programmed Cas9 (D10A/H840A) and protospacer 4 target DNA duplexes [same as in (B)] containing WT and mutated PAM motifs. The Cas9 (D10A/H840A)–RNA complex was titrated from 100 pM to 1 μM.

  • Fig. 5

    Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features. (A) (Top) In type II CRISPR/Cas systems, Cas9 is guided by a two-RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA (see fig. S1). (Bottom) A chimeric RNA generated by fusing the 3′ end of crRNA to the 5′ end of tracrRNA. (B) A plasmid harboring protospacer 4 target sequence and a WT PAM was subjected to cleavage by Cas9 programmed with tracrRNA(4-89):crRNA-sp4 duplex or in vitro–transcribed chimeric RNAs constructed by joining the 3′ end of crRNA to the 5′ end of tracrRNA with a GAAA tetraloop. Cleavage reactions were analyzed by restriction mapping with XmnI. Sequences of chimeric RNAs A and B are shown with DNA-targeting (yellow), crRNA repeat-derived sequences (orange), and tracrRNA-derived (light blue) sequences. (C) Protospacer 4 DNA duplex cleavage reactions were performed as in Fig. 1B. (D) Five chimeric RNAs designed to target the GFP gene were used to program Cas9 to cleave a GFP gene–containing plasmid. Plasmid cleavage reactions were performed as in Fig. 3E, except that the plasmid DNA was restriction mapped with AvrII after Cas9 cleavage.

Additional Files

  • A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

    Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier

    Materials/Methods, Supporting Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Tables S1 to S3
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    Revised 15 August 2012: Formatting errors and typos have been corrected. Additionally, the format of the tables has been revised, and a duplicate entry has been removed from table S2.
    The original version is still available here.

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