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. 2012 May 22;109(21):8061-6.
doi: 10.1073/pnas.1117984109. Epub 2012 May 7.

Monomeric site-specific nucleases for genome editing

Benjamin P Kleinstiver  1 , Jason M Wolfs, Tomasz Kolaczyk, Alanna K Roberts, Sherry X Hu, David R Edgell
Affiliations

Monomeric site-specific nucleases for genome editing

Benjamin P Kleinstiver et al. Proc Natl Acad Sci U S A. .

Abstract

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5'-CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5'-CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Design and functionality of Tev-ZFEs. (A) Modeling of a Tev-zinc finger fusion with DNA substrate (light green) using structures of the I-TevI catalytic domain in green (PDB 1MK0), the I-TevI DNA-binding domain cocrystal in blue (PDB 1I3J), and the Zif268 cocrystal in red (PDB 1AAY). (B) The TZ-ryA substrate is colored according to the structural model. Shown is the top strand of the I-TevI td homing site substrate fused to the 5′ end of the ryA-binding site for all wild-type substrates tested. The substrate is numbered from the first base of the td homing site sequence (the numbering scheme is reverse of that used for the native td homing site). The substrates tested differ by insertion or deletion of td sequence at the junction of the td/ryA sites. (C) Percent survival of three representative Tev-ryA ZFEs in the bacterial two-plasmid selection. All Tev-ryA ZFEs were tested against plasmids containing various length substrates (TZ1.30–1.38), plasmids lacking a target site (p11lacY), and TZ1.33 plasmids with single or double mutations in the CNNNG motif (G5A and C1A/G5A) (Table S1). (D) Percent survival of TevN201-ryA and TevN201-ryB ZFEs on their cognate and reciprocal target sites. Data are plotted with SD for n ≥ 3.
Fig. 2.
Fig. 2.
TevN201-ZFE is a monomer with a preferred cleavage site. (A) (Left) Plot of initial reaction progress for seven TevN201-ZFE concentrations expressed as percent linear product. Protein concentrations from highest to lowest are 47 nM, 32.5 nM, 23 nM, 11 nM, 6 nM, 3 nM, and 0.7 nM. (Right) Graph of initial reaction velocity (nM⋅s−1) versus TevN201-ZFE concentration (nM). (B) Graphic representation of cleavage assays with 90 nM TevN201-ZFE and 10 nM one- or two-site TZ1.33 plasmids (Left and Right, respectively). The two-site plasmid had the TZ-ryA sites in the opposite (shown) or same (Fig. S2B) orientation. FLL, full-length linear; L1+L2, linear products; OC, open-circle (nicked); SC, supercoiled. (C) Mapping of TevN201-ZFE cleavage sites on the TZ1.33 substrate, with top and bottom cleavage sites indicated below on the TZ-ryA substrate by open and closed triangles, respectively. (D) Activity of TevN201-ZFE on the wild-type TZ1.33, or the TZ1.33 G5A and TZ1.33 C1A/G5A mutant substrates. A graph of EC0.5max determinations for each substrate is shown to the right, with EC0.5max values in nanomolars. Data are plotted as averages of three independent replicates with SDs.
Fig. 3.
Fig. 3.
Tev-ZFEs can induce recombination in a eukaryotic system. Shown are normalized β-galactosidase units from a yeast-based recombination assay for the indicated nuclease/substrate combinations. Activity was normalized to a homodimeric FokI-Zif268 ZFN-positive control. Data are plotted with SD for n = 4.
Fig. 4.
Fig. 4.
Design and functionality of Tev-LHEs. (A) Modeling of a Tev-Onu E1 fusion with DNA substrate (light green) using structures of the I-TevI catalytic domain in green (PDB 1MK0), the I-TevI DNA-binding domain cocrystal in blue (PDB 1I3J), and the I-OnuI cocrystal in red (PDB 3QQY). Shown are fusion points at which the I-TevI fragment has been shortened. (B) The Tev-Onu E1 (TO) substrate is colored according to the structural model. Shown is the top strand of the I-TevI td homing site substrate fused to the 5′ end of the Onu E1-binding site. The substrates are numbered from the first base of the td homing site sequence and differ by the deletion of td nucleotides at the junction of the td/Onu E1 sites. (C) Percent survival of Tev-LHEs in the bacterial two-plasmid selection with various length target sites (TO1.12–1.30). All Tev-LHEs tested were in the I-OnuI E1 E22Q background. (D) Percent survival of TevR27A(N201G4)-OnuE1 and TevR27A(N201G4)-OnuE1(E22Q) on TO1.30, TO1.30G5A, and TZ1.33. Data are plotted with SD for n = 3.
Fig. 5.
Fig. 5.
Cleavage requirements do not limit GIY-ZFE and GIY-LHE applicability. (A) A diverse set of monomeric and sequence specific reagents can be generated by fusing distinct GIY-YIG domain linker lengths to engineered DNA-binding platforms, including zinc-finger arrays and inactive LAGLIDADGs. (B) Shown is the distribution of the CNNNG motif in a 35-bp window flanking 8,829 predicted ZFN sites on zebrafish chromosome 1. The number of occurrences of the “C” of the motif at each distance is indicated. (C) Unique ZFN sites were grouped according to the number of occurrences of the CNNNG motif in the 35-bp window. The red line is the expected number of ZFN sites for each group based on a binomial distribution.
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