- Which one of the following statements regarding use of hybrid nucleases for plant genome engineering is INCORRECT?
(1) For gene knock out experiments, the nuclease is expressed without a donor DNA template.
(2) Hybrid nucleases typically comprise two protein subunits that dimerize at their nuclease domain.
(3) Zinc finger nucleases (ZFNs) can efficiently target all nuclear genes with equal efficiency.
(4) Both ZFNs and TALENs are fused with cleavage domains of Fokl endonuclease.The incorrect statement is (3) Zinc finger nucleases (ZFNs) can efficiently target all nuclear genes with equal efficiency.
Option-by-option explanation
(1) “For gene knock out experiments, the nuclease is expressed without a donor DNA template.” – Correct
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Gene knockouts with hybrid nucleases (ZFNs, TALENs, CRISPR/Cas) typically rely on non‑homologous end joining (NHEJ).
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The nuclease creates a double‑strand break; error‑prone NHEJ repairs it, introducing small insertions/deletions that disrupt the coding sequence.
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No donor template is required when the goal is simply to knock out the gene.
(2) “Hybrid nucleases typically comprise two protein subunits that dimerize at their nuclease domain.” – Correct
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Classical engineered nucleases like ZFNs and TALENs are built from:
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A programmable DNA‑binding domain (zinc fingers or TALE repeats).
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A FokI nuclease domain, which must dimerize to cut DNA.
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Two monomers bind opposite strands with proper spacing; their FokI domains dimerize to cleave.
(3) “Zinc finger nucleases (ZFNs) can efficiently target all nuclear genes with equal efficiency.” – Incorrect
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ZFN design is sequence‑context dependent:
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Not all triplet combinations are equally well supported by zinc‑finger modules.
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Some target sites are hard or impossible to design with high affinity and specificity.
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Chromatin accessibility and local DNA structure further affect cutting efficiency.
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Thus ZFNs do not target all nuclear genes equally well, and some loci are inefficient or off‑target‑prone.
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This blanket statement of universal, equal efficiency is therefore incorrect.
(4) “Both ZFNs and TALENs are fused with cleavage domains of FokI endonuclease.” – Correct
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Both platforms use the same strategy: customizable DNA binding domain (ZF or TALE) fused to the FokI catalytic domain.
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FokI provides the nonspecific cleavage function; specificity comes from the attached DNA‑binding protein.
Because statement (3) is the only one that is factually wrong, the correct answer is option (3).
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