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. 2014 May 9;289(19):13284-94.
doi: 10.1074/jbc.M113.539726. Epub 2014 Mar 14.

Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease

Hongfan Chen  1 , Jihoon Choi, Scott Bailey
Affiliations

Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease

Hongfan Chen et al. J Biol Chem. .

Abstract

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and _target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA _target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Keywords: CRISPR; Cas9; DNA; DNA Transformation; DNA-binding Protein; Genome Editing; Nuclease; RNA; RNA-binding Protein.

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Figures

FIGURE 1.
FIGURE 1.
The type II CRISPR-Cas system of S. thermophilus LMG18311. A, Coomassie Blue-stained SDS-polyacrylamide gel of Cas9, Cas9 D9A, Cas9 D599A, and Cas9 D9A,D599A. B, logo plot revealing the PAM for LMG18311 Cas9. The positions of the protospacer, PAM, and linker are indicated. C, schematic representation of the crRNA (green), tracrRNA (blue), and DNA _target (black). The positions of the protospacer, PAM, and linker are indicated. The site at which the crRNA and tracrRNA are fused to generate the sgRNA is indicated with a dotted line.
FIGURE 2.
FIGURE 2.
LMG18311 Cas9 and cognate sgRNA can provide resistance to plasmid transformation in E. coli. A, schematic representation of transformation assay. B, interference of plasmid transformation by LMG18311 Cas9 and sgRNA in E. coli cells. Transformation efficiency is expressed as cfu per 5 ng of plasmid DNA. Average values from at least three biological replicates are shown, with error bars representing 1 S.D. C, effect of mutation in the PAM sequence on plasmid transformation efficiency. D, effect of linker length on plasmid transformation efficiency. PS denotes the protospacer sequence.
FIGURE 3.
FIGURE 3.
DNA cleavage by LMG18311 Cas9 in vitro. A, RNA-guided cleavage by Cas9. Reaction mixtures containing 5 nm _target plasmid, 25 nm Cas9, 25 nm crRNA, 25 nm tracrRNA, and 10 mm Mg2+ were incubated for 30 min at 37 °C. B, a cognate sgRNA can substitute for crRNA and tracrRNA. C, cleavage of a plasmid _target is dictated by the sgRNA sequence. D, cleavage of a plasmid _target by active site mutants of Cas9. E, cleavage of a synthetic dsDNA by active site mutants of Cas9. The dsDNA was radiolabeled at the 5′ end of the complementary strand (left) or the noncomplementary strand (right). Reactions were performed as in A, and products were separated by 10% denaturing PAGE. The cleavage sites are indicated with arrows in the schematic diagram (bottom). 50 nt, 50 nucleotides; 37 nt, 37 nucleotides. F, cleavage of plasmid _targets containing mutations in the PAM sequence. G, cleavage of plasmid _targets containing the indicated linker lengths. Average values from at least three biological replicates are shown, with error bars representing 1 S.D. In A–C and E–F, the positions of negatively supercoiled (nSC), linear (L) and nicked or open circle (OC) plasmid are indicated. The linear control is a digestion of the plasmid _target with the restriction enzyme AgeI.
FIGURE 4.
FIGURE 4.
Metal dependence of DNA cleavage by Cas9. A, cleavage of a _target plasmid by Cas9 with either no metal or 1 mm of the indicated metal ions. All reactions were treated with 0.5 mm EDTA prior to metal addition. B, cleavage of a _target plasmid by active site mutants of Cas9 in the presence of 10 mm Ca2+. In both panels, the positions of negatively supercoiled (nSC), linear (L), and nicked or open circle (OC) plasmid are indicated. The linear control is a digestion of the plasmid _target with the restriction enzyme AgeI.
FIGURE 5.
FIGURE 5.
DNA _target binding by Cas9. A, a representative gel shift assay for Cas9-sgRNA and the binding curve measured from the assay. B and C, bar graph plotting Kd values for DNA _targets with PAM mutations (labeled red) (B) or DNA _targets with different linker lengths (labeled red) (C). Average values from at least three replicates are shown, with error bars representing 1 S.D. _targets where binding was not observed are shown with Kd values at the lower limit (> 1000 nm). PS denotes the protospacer sequence.
FIGURE 6.
FIGURE 6.
Mapping the Cas9 cleavage sites in plasmid _targets with different linker lengths. A and B, direct sequencing electropherograms for plasmid-sp1 (A) and plasmid-sp2 (B) from complementary strand primer (bottom left) and noncomplementary strand primer (bottom right) are shown. Termination of primer extension in the sequencing reactions reveals the positions of the cleavage site (red lines). The positions of the protospacer, PAM, and linker are indicated. The 3′-terminal A addition, indicated by asterisk, is an artifact of the sequencing reaction.
FIGURE 7.
FIGURE 7.
Schematic representation of cut site selection by the HNH and RuvC-like domains of Cas9. Top, a schematic with a native DNA _target; bottom, a schematic with a DNA _target containing a longer length linker. Cleavage sites on the complementary and noncomplementary strands are indicated by yellow arrows. For clarity, the tracrRNA is not shown.
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References

    1. Mali P., Yang L., Esvelt K. M., Aach J., Guell M., DiCarlo J. E., Norville J. E., Church G. M. (2013) RNA-guided human genome engineering via Cas9. Science 339, 823–826 - PMC - PubMed
    1. Cong L., Ran F. A., Cox D., Lin S., Barretto R., Habib N., Hsu P. D., Wu X., Jiang W., Marraffini L. A., Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 - PMC - PubMed
    1. Jinek M., East A., Cheng A., Lin S., Ma E., Doudna J. (2013) RNA-programmed genome editing in human cells. Elife 2, e00471–e00471 - PMC - PubMed
    1. Cho S. W., Kim S., Kim J. M., Kim J.-S. (2013) _targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 - PubMed
    1. Shen B., Zhang J., Wu H., Wang J., Ma K., Li Z., Zhang X., Zhang P., Huang X. (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene _targeting. Cell Res. 23, 720–723 - PMC - PubMed

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