Zinc Finger Nuclease DNA Cleavage

Target Geometry

Spacer length 6 bp

Search (1D diffusion) speed 1.0x

FokI Nuclease Variant

Reaction State

Current phase

Searching…

FokI geometry match

On-target cuts

Off-target cuts

Attempt time

0.0s

How it works

A zinc finger nuclease (ZFN) monomer fuses a Cys2His2 zinc finger array (each finger reads ~3 bp in the DNA major groove) to the non-specific cleavage domain of the restriction enzyme FokI. FokI's nuclease domain is catalytically dead as a monomer — cutting requires two FokI domains to dimerize.

ZFNs are therefore always used in engineered pairs: one monomer binds a "left" half-site, the other binds a "right" half-site on the opposite strand in tail-to-tail orientation, separated by an unspecified spacer. Only when both are bound does the tethered FokI pair sit close enough to dimerize and cut both strands, leaving a staggered double-strand break with 4-nt 5′ overhangs.

Cleavage needs:  binding(left) AND binding(right) AND dimer(FokI, FokI)
Geometry match:  M(s) = exp( -(s - 6)^2 / (2·2.2^2) )   s = spacer length, bp
Cleavage prob. per attempt ≈ M(s)  (Bitinaite et al. 1998; Smith et al. 2000)

Spacer length sets the geometry: FokI's dimer interface only forms efficiently around 5–6 bp of spacer, modeled above as a Gaussian falloff. Too short or too long and binding still happens, but the catalytic domains can't reach each other, so dimerization keeps failing and the pair releases.

Early ZFNs used wild-type FokI, whose domains can dimerize with any nearby FokI copy — including two copies of the same monomer at a look-alike site elsewhere — causing off-target double-strand breaks (flashed in red on the flanking DNA here). Engineered obligate heterodimer variants (Miller et al. 2007; Szczepek et al. 2007) add complementary charge mutations at the dimer interface so only the intended left+right pair can dimerize, eliminating that homodimer route.

Left ZFN monomer Right ZFN monomer FokI cleavage domain Spacer (uncut sequence)

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