Originally published 28 June 2012; corrected 15 August 2012

http:///cgi/content/full/science.1225829/DC1

Supplementary Materials for

A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive

Bacterial Immunity

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

Emmanuelle Charpentier*

*To whom correspondence should be addressed. E-mail: doudna@berkeley.edu (J.A.D.);

emmanuelle.charpentier@mims.umu.se (E.C.)

Published 28 June 2012 on Science Express

DOI: 10.1126/science.1225829

This PDF file includes:

Materials and Methods

Figs. S1 to S15

Tables S1 to S3

Full Reference List

Correction: 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.

SUPPLEMENTARY MATERIALS AND METHODS

Bacterial strains and culture conditions. Table S1 lists the bacterial strains used in the study. Streptococcus pyogenes, cultured in THY medium (Todd Hewitt Broth (THB, Bacto, Becton Dickinson) supplemented with 0.2% yeast extract (Oxoid)) or on TSA (trypticase soy agar, BBL, Becton Dickinson) supplemented with 3% sheep blood, was incubated at 37°C in an atmosphere supplemented with 5% CO2 without shaking. Escherichia coli, cultured in Luria-Bertani (LB) medium and agar, was incubated at 37°C with shaking. When required, suitable antibiotics were added to the medium at the following final concentrations: ampicillin, 100 µg/ml for E. coli; chloramphenicol, 33 µg/ml for E. coli; kanamycin, 25 µg/ml for E. coli and 300 µg/ml for S. pyogenes. Bacterial cell growth was monitored periodically by measuring the optical density of culture aliquots at 620 nm using a microplate reader (SLT Spectra Reader).

Transformation of bacterial cells. Plasmid DNA transformation into E. coli cells was performed according to a standard heat shock protocol (39). Transformation of S. pyogenes was performed as previously described with some modifications (40). The transformation assay performed to monitor in vivo CRISPR/Cas activity on plasmid maintenance was essentially carried out as described previously (4). Briefly, electro-competent cells of S. pyogenes were equalized to the same cell density and electroporated with 500 ng of plasmid DNA. Every transformation was plated two to three times and the experiment was performed three times independently with different batches of competent cells for statistical analysis. Transformation efficiencies were calculated as CFU (colony forming units) per µg of DNA. Control transformations were performed with sterile water and backbone vector pEC85.

DNA manipulations. DNA manipulations including DNA preparation, amplification, digestion, ligation, purification, agarose gel electrophoresis were performed according to standard techniques (39) with minor modifications. Protospacer plasmids for the in vitro cleavage and S. pyogenes transformation assays were constructed as described previously (4). Additional pUC19-based protospacer plasmids for in vitro cleavage assays were generated by ligating annealed oligonucleotides between digested EcoRI and BamHI sites in pUC19. The GFP gene-containing plasmid has been described previously (41). Kits (Qiagen) were used for DNA purification and plasmid preparation. Plasmid mutagenesis was performed using QuikChange® II XL kit (Stratagene) or QuikChange site-directed mutagenesis kit (Agilent). All plasmids used in this study were sequenced at LGC Genomics or the UC Berkeley DNA Sequencing Facility and are listed in Table S2. VBC-Biotech Services, Sigma-Aldrich and Integrated DNA Technologies supplied the synthetic oligonucleotides and RNAs listed in Table S3.

In vitro transcription and purification of RNA. RNA was in vitro transcribed using T7 Flash in vitro Transcription Kit (Epicentre, Illumina company) and PCR-generated DNA templates carrying a T7 promoter sequence. RNA was gel-purified and quality-checked prior to use. The primers used for the preparation of RNA templates from S. pyogenes SF370, Listeria innocua Clip 11262 and Neisseria meningitidis A Z2491 are listed in Table S3.

Protein purification. The sequence encoding Cas9 (residues 1-1368) was PCR-amplified from the genomic DNA of S. pyogenes SF370 and inserted into a custom pET-based expression vector using ligation-independent cloning (LIC). The resulting fusion construct contained an N-terminal hexahistidine-maltose binding protein (His6-MBP) tag, followed by a peptide sequence containing a tobacco etch virus (TEV) protease cleavage site. The protein was expressed in E. coli strain BL21 Rosetta 2 (DE3) (EMD Biosciences), grown in 2xTY medium at 18°C for 16 h following induction with 0.2 mM IPTG. The protein was purified by a combination of affinity, ion exchange and size exclusion chromatographic steps. Briefly, cells were lysed in 20 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP (supplemented with protease inhibitor cocktail (Roche)) in a homogenizer (Avestin). Clarified lysate was bound in batch to Ni-NTA agarose (Qiagen). The resin was washed extensively with 20 mM Tris pH 8.0, 500 mM NaCl and the bound protein was eluted in 20 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol. The His6-MBP affinity tag was removed by cleavage with TEV protease, while the protein was dialyzed overnight against 20 mM HEPES pH 7.5, 150 mM KCl, 1 mM TCEP, 10% glycerol. The cleaved Cas9 protein was separated from the fusion tag by purification on a 5 ml SP Sepharose HiTrap column (GE Life Sciences), eluting with a linear gradient of 100 mM – 1 M KCl. The protein was further purified by size exclusion chromatography on a Superdex 200 16/60 column in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP. Eluted protein was concentrated to ~8 mg.ml-1, flash-frozen in liquid nitrogen and stored at -80°C. Cas9 D10A, H840A and D10A/H840A point mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent) and confirmed by DNA sequencing. The proteins were purified following the same procedure as for the wild-type Cas9 protein.

Cas9 orthologs from Streptococcus thermophilus (LMD-9,YP_820832.1), L.

innocua (Clip11262, NP_472073.1), Campylobacter jejuni (subsp. jejuni NCTC 11168, YP_002344900.1) and N. meningitidis (Z2491, YP_002342100.1) were expressed in BL21 Rosetta (DE3) pLysS cells (Novagen) as His6-MBP (N. meningitidis and C. jejuni), His6-Thioredoxin (L. innocua) and His6-GST (S. thermophilus) fusion proteins, and purified essentially as for S. pyogenes Cas9 with the following modifications. Due to large amounts of co-purifying nucleic acids, all four Cas9 proteins were purified by an additional heparin sepharose step prior to gel filtration, eluting the bound protein with a linear gradient of 100 mM – 2 M KCl. This successfully removed nucleic acid contamination from the C. jejuni, N. meningitidis and L. innocua proteins, but failed to remove co-purifying nucleic acids from the S. thermophilus Cas9 preparation. All proteins were concentrated to 1-8 mg.ml-1 in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP, flash-frozen in liquid N2 and stored at -80°C.

Plasmid DNA cleavage assay. Synthetic or in vitro-transcribed tracrRNA and crRNA were pre-annealed prior to the reaction by heating to 95°C and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) was incubated for 60 min at 37°C with purified Cas9 protein (50-500 nM) and tracrRNA:crRNA duplex (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl2. The reactions were stopped with 5X DNA loading buffer containing 250 mM EDTA, resolved by 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide

staining. For the Cas9 mutant cleavage assays, the reactions were stopped with 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior to loading on the agarose gel.

Metal-dependent cleavage assay. Protospacer 2 plasmid DNA (5 nM) was incubated for 1 h at 37°C with Cas9 (50 nM) pre-incubated with 50 nM tracrRNA:crRNA-sp2 in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) supplemented with 1, 5 or 10 mM MgCl2, 1 or 10 mM of MnCl2, CaCl2, ZnCl2, CoCl2, NiSO4 or CuSO4. The reaction was stopped by adding 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.

Single-turnover assay. Cas9 (25 nM) was pre-incubated 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with duplexed tracrRNA:crRNA-sp2 (25 nM, 1:1) or both RNAs (25 nM) not pre-annealed and the reaction was started by adding protospacer 2 plasmid DNA (5 nM). The reaction mix was incubated at 37°C. At defined time intervals, samples were withdrawn from the reaction, 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) was added to stop the reaction and the cleavage was monitored by 1% agarose gel electrophoresis and ethidium bromide staining. The same was done for the single turnover kinetics without pre-incubation of Cas9 and RNA, where protospacer 2 plasmid DNA (5 nM) was mixed in cleavage buffer with duplex tracrRNA:crRNA-sp2 (25 nM) or both RNAs (25 nM) not pre-annealed, and the reaction was started by addition of Cas9 (25 nM). Percentage of cleavage was analyzed by densitometry and the average of three independent experiments was plotted against time. The data were fit by nonlinear regression analysis and the cleavage rates (kobs [min-1]) were calculated.

Multiple-turnover assay. Cas9 (1 nM) was pre-incubated for 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with pre-annealed tracrRNA:crRNA-sp2 (1 nM, 1:1). The reaction was started by addition of protospacer 2 plasmid DNA (5 nM). At defined time intervals, samples were withdrawn and the reaction was stopped by adding 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA). The cleavage reaction was resolved by 1% agarose gel electrophoresis, stained with ethidium bromide and the percentage of cleavage was analyzed by densitometry. The results of four independent experiments were plotted against time (min).

Oligonucleotide DNA cleavage assay. DNA oligonucleotides (10 pmol) were radiolabeled by incubating with 5 units T4 polynucleotide kinase (New England Biolabs) and ~3–6 pmol (~20–40 mCi) [γ-32P]-ATP (Promega) in 1X T4 polynucleotide kinase reaction buffer at 37°C for 30 min, in a 50 μL reaction. After heat inactivation (65°C for 20 min), reactions were purified through an Illustra MicroSpin G-25 column (GE Healthcare) to remove unincorporated label. Duplex substrates (100 nM) were generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95°C for 3 min, followed by slow cooling to room temperature. For cleavage assays, tracrRNA and crRNA were annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) was

pre-incubated with the annealed tracrRNA:crRNA duplex (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions were initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37°C. Reactions were quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products were resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging (Storm, GE Life Sciences). Cleavage assays testing PAM requirements (Fig. 4B) were carried out using DNA duplex substrates that had been pre-annealed and purified on an 8% native acrylamide gel, and subsequently radiolabeled at both 5’ ends. The reactions were set-up and analyzed as above.

Electrophoretic mobility shift assays. Target DNA duplexes were formed by mixing of each strand (10 nmol) in deionized water, heating to 95°C for 3 min and slow cooling to room temperature. All DNAs were purified on 8% native gels containing 1X TBE. DNA bands were visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H2O. Eluted DNA was ethanol precipitated and dissolved in DEPC-treated H2O. DNA samples were 5’ end labeled with [γ-32P]-ATP using T4 polynucleotide kinase (New England Biolabs) for 30 min at 37°C. PNK was heat denatured at 65°C for 20 min, and unincorporated radiolabel was removed using an Illustra MicroSpin G-25 column (GE Healthcare). Binding assays were performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 D10A/H840A double mutant was programmed with equimolar amounts of pre-annealed tracrRNA:crRNA duplex and titrated from 100 pM to 1 μM. Radiolabeled DNA was added to a final concentration of 20 pM. Samples were incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing 1X TBE and 5 mM MgCl2. Gels were dried and DNA visualized by phosphorimaging.

In silico analysis of DNA and protein sequences. Vector NTI package (Invitrogen) was used for DNA sequence analysis (Vector NTI) and comparative sequence analysis of proteins (AlignX).

In silico modeling of RNA structure and co-folding. In silico predictions were performed using the Vienna RNA package algorithms (42, 43). RNA secondary structures and co-folding models were predicted with RNAfold and RNAcofold, respectively and visualized with VARNA (44).

SUPPLEMENTARY FIGURES

Fig. S1. The type II RNA-mediated CRISPR/Cas immune pathway. The

expression and interference steps are represented in the drawing. The type II

CRISPR/Cas loci are composed of an operon of four genes (blue) encoding the

proteins Cas9, Cas1, Cas2 and Csn2, a CRISPR array consisting of a leader

sequence followed by identical repeats (black rectangles) interspersed with

unique genome-targeting spacers (colored diamonds) and a sequence encoding

the trans-activating tracrRNA (red). Represented here is the type II CRISPR/Cas

locus of S. pyogenes SF370 (Accession number NC_002737) (4). Experimentally

confirmed promoters and transcriptional terminator in this locus are indicated (4). molecule that undergoes a maturation process specific to the type II systems (4).

In S. pyogenes SF370, tracrRNA is transcribed as two primary transcripts of 171

and 89 nt in length that have complementarity to each repeat of the pre-crRNA.

The first processing event involves pairing of tracrRNA to pre-crRNA, forming a

duplex RNA that is recognized and cleaved by the housekeeping endoribonuclease RNase III (orange) in the presence of the Cas9 protein. RNase

III-mediated cleavage of the duplex RNA generates a 75-nt processed tracrRNA

and a 66-nt intermediate crRNAs consisting of a central region containing a

sequence of one spacer, flanked by portions of the repeat sequence. A second

processing event, mediated by unknown ribonuclease(s), leads to the formation

of mature crRNAs of 39 to 42 nt in length consisting of 5’-terminal spacer-derived

guide sequence and repeat-derived 3’-terminal sequence. Following the first and

second processing events, mature tracrRNA remains paired to the mature

crRNAs and bound to the Cas9 protein. In this ternary complex, the dual

tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9

to the cognate target DNA. Target recognition by the Cas9-tracrRNA:crRNA

complex is initiated by scanning the invading DNA molecule for homology

between the protospacer sequence in the target DNA and the spacer-derived

sequence in the crRNA. In addition to the DNA protospacer-crRNA spacer

complementarity, DNA targeting requires the presence of a short motif (NGG,

where N can be any nucleotide) adjacent to the protospacer (protospacer

adjacent motif - PAM). Following pairing between the dual-RNA and the

protospacer sequence, an R-loop is formed and Cas9 subsequently introduces a

double-stranded break (DSB) in the DNA. Cleavage of target DNA by Cas9

requires two catalytic domains in the protein. At a specific site relative to the

PAM, the HNH domain cleaves the complementary strand of the DNA while the

RuvC-like domain cleaves the noncomplementary strand.

Fig. S2. Purification of Cas9 nucleases. (A) S. pyogenes Cas9 was expressed

in E. coli as a fusion protein containing an N-terminal His6-MBP tag and purified

by a combination of affinity, ion exchange and size exclusion chromatographic

steps. The affinity tag was removed by TEV protease cleavage following the

affinity purification step. Shown is a chromatogram of the final size exclusion

chromatography step on a Superdex 200 (16/60) column. Cas9 elutes as a single

monomeric peak devoid of contaminating nucleic acids, as judged by the ratio of

absorbances at 280 and 260 nm. Inset; eluted fractions were resolved by SDS-

PAGE on a 10% polyacrylamide gel and stained with SimplyBlue Safe Stain

(Invitrogen). (B) SDS-PAGE analysis of purified Cas9 orthologs. Cas9 orthologs

were purified as described in Supplementary Materials and Methods. 2.5 μg of

each purified Cas9 were analyzed on a 4-20% gradient polyacrylamide gel and

stained with SimplyBlue Safe Stain.

Fig. S3. Cas9 guided by dual-tracrRNA:crRNA cleaves protospacer plasmid

and oligonucleotide DNA. See Fig. 1. The protospacer 1 sequence originates

from S. pyogenes SF370 (M1) SPy_0700, target of S. pyogenes SF370 crRNA-

sp1 (4). Here, the protospacer 1 sequence was manipulated by changing the

PAM from a nonfunctional sequence (TTG) to a functional one (TGG). The

protospacer 4 sequence originates from S. pyogenes MGAS10750 (M4)

MGAS10750_Spy1285, target of S. pyogenes SF370 crRNA-sp4 (4). (A)

Protospacer 1 plasmid DNA cleavage guided by cognate tracrRNA:crRNA

duplexes. The cleavage products were resolved by agarose gel electrophoresis

and visualized by ethidium bromide staining. M, DNA marker; fragment sizes in

base pairs are indicated. (B) Protospacer 1 oligonucleotide DNA cleavage guided

by cognate tracrRNA:crRNA-sp1 duplex. The cleavage products were resolved

by denaturating polyacrylamide gel electrophoresis and visualized by phosphorimaging. Fragment sizes in nucleotides are indicated. (C) Protospacer 4

oligonucleotide DNA cleavage guided by cognate tracrRNA:crRNA-sp4 duplex.

The cleavage products were resolved by denaturating polyacrylamide gel

electrophoresis and visualized by phosphorimaging. Fragment sizes in

nucleotides are indicated. (A, B, C) Experiments in (A) were performed as in Fig.

1A; in (B) and in (C) as in Fig. 1B. (B, C) A schematic of the tracrRNA:crRNA-

target DNA interaction is shown below. The regions of crRNA complementarity to

tracrRNA and the protospacer DNA are represented in orange and yellow,

respectively. The PAM sequence is indicated in grey.

Fig. S4. Cas9 is a Mg2+-dependent endonuclease with 3’-5’ exonuclease

activity. See Fig. 1. (A) Protospacer 2 plasmid DNA was incubated with Cas9

complexed with tracrRNA:crRNA-sp2 in the presence of different concentrations

of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, Ni2+ or Cu2+. The cleavage products were

resolved by agarose gel electrophoresis and visualized by ethidium bromide

staining. Plasmid forms are indicated. (B) A protospacer 4 oligonucleotide DNA

duplex containing a PAM motif was annealed and gel-purified prior to

radiolabeling at both 5’ ends. The duplex (10 nM final concentration) was

incubated with Cas9 programmed with tracrRNA (nucleotides 23-89) and crRNA-

sp4 (500 nM final concentration, 1:1). At indicated time points (min), 10 μl

aliquots of the cleavage reaction were quenched with formamide buffer

containing 0.025% SDS and 5 mM EDTA, and analyzed by denaturing

polyacrylamide gel electrophoresis as in Fig. 1B. Sizes in nucleotides are indicated.

Fig. S5. Dual-tracrRNA:crRNA directed Cas9 cleavage of target DNA is site-specific. See Fig. 1 and fig. S3. (A) Mapping of protospacer 1 plasmid DNA cleavage. Cleavage products from fig. S3A were analyzed by sequencing as in Fig. 1C. Note that the 3’ terminal A overhang (asterisk) is an artifact of the sequencing reaction. (B) Mapping of protospacer 4 oligonucleotide DNA cleavage. Cleavage products from fig. S3C were analyzed by denaturing polyacrylamide gel electrophoresis alongside 5’ end-labeled oligonucleotide size markers derived from the complementary and noncomplementary strands of the protospacer 4 duplex DNA. M, marker; P, cleavage product. Fragment sizes in nucleotides are indicated. (C) Schematic representations of tracrRNA, crRNA-sp1 and protospacer 1 DNA sequences (left) and tracrRNA, crRNA-sp4 and protospacer 4 DNA sequences (right). tracrRNA:crRNA forms a dual-RNA structure directed to complementary protospacer DNA through crRNA-protospacer DNA pairing. The regions of crRNA complementary to tracrRNA and the protospacer DNA are represented in orange and yellow, respectively. The cleavage sites in the complementary and noncomplementary DNA strands mapped in (A) (left) and (B) (right) are represented with red arrows (A and B, complementary strand) and a red thick line (B, noncomplementary strand) above the sequences, respectively.

Fig. S6. Dual-tracrRNA:crRNA directed Cas9 cleavage of target DNA is fast

and efficient. See Fig. 1. (A) Single turnover kinetics of Cas9 under different

RNA pre-annealing and protein-RNA pre-incubation conditions. Protospacer 2

plasmid DNA was incubated with either Cas9 pre-incubated with pre-annealed

tracrRNA:crRNA-sp2 (○), Cas9 not pre-incubated with pre-annealed tracrRNA:crRNA-sp2 (●), Cas9 pre-incubated with not pre-annealed tracrRNA

and crRNA-sp2 (□) or Cas9 not pre-incubated with not pre-annealed RNAs (■).

The cleavage activity was monitored in a time-dependent manner and analyzed

by agarose gel electrophoresis followed by ethidium bromide staining. The

average percentage of cleavage from three independent experiments is plotted

against the time (min) and fitted with a nonlinear regression. The calculated

cleavage rates (kobs) are shown in the table on the right. The results suggest that

the binding of Cas9 to the RNAs is not rate-limiting under the conditions tested.

Plasmid forms are indicated. The obtained kobs values are comparable to those of

restriction endonucleases which are typically of the order of 1-10 min-1 (45-47).

(B) Cas9 is a multiple turnover endonuclease. Cas9 loaded with duplexed

tracrRNA:crRNA-sp2 (1 nM, 1:1:1 – indicated with red line on the graph) was

incubated with a 5-fold excess of native protospacer 2 plasmid DNA. Cleavage

was monitored by withdrawing samples from the reaction at defined time intervals

(0 to 120 min) followed by agarose gel electrophoresis analysis (top) and

determination of cleavage product amount (nM) (bottom). Standard deviations of

three independent experiments are indicated. In the time interval investigated, 1

nM Cas9 was able to cleave ~2.5 nM plasmid DNA.

Fig. S7. Cas9 contains two nuclease domains: HNH (McrA-like) and RuvC-

like (N-terminal RNase H fold) resolvase. See Fig. 2. The amino-acid

sequence of Cas9 from S. pyogenes SF370 (Accession number NC_002737) is

represented. The three predicted RuvC-like motifs and the predicted HNH motif

are shaded light blue and purple, respectively. Residues Asp10 and His840,

which were substituted by Ala in this study are shown in red and highlighted by a

red asterisk above the sequence. Underlined residues are highly conserved

among Cas9 proteins from different species. Note that in a previous study and

based on computational predictions, Makarova et al. (23) predicted coupling of

the two nuclease-like activities, which is now confirmed experimentally in the

present study (Fig. 2 and fig. S8).

Fig. S8. The HNH and RuvC-like domains of Cas9 direct cleavage of the

complementary and noncomplementary DNA strand, respectively. See Fig.

2. Protospacer DNA cleavage by cognate tracrRNA:crRNA-directed Cas9

mutants containing mutations in the HNH or RuvC-like domain. (A) Protospacer 1

plasmid DNA cleavage. The experiment was performed as in Fig. 2A. Plasmid

DNA conformations and sizes in base pairs are indicated. (B) Protospacer 4

oligonucleotide DNA cleavage. The experiment was performed as in Fig. 2B.

Sizes in nucleotides are indicated.