#zinc finger nucleases

Transgenic Plant -2

ZFNs induce genome modification by creating:

Zinc finger nucleases induce targeted genome modification by introducing double-strand breaks which are highly recombinogenic lesions activating cellular DNA damage response. After FokI dimer cleavage within spacer producing 5' overhangs, MRN complex Mre11-Rad50-Nbs1 senses ends, recruits ATM kinase phosphorylating H2AX, mediators 53BP1, initiating cell-cycle checkpoint. Breaks must be repaired to avoid apoptosis. In absence of donor, classical NHEJ mediated by Ku70/Ku80 heterodimer binding ends, DNA-PKcs recruitment, Artemis processing and Lig4-XRCC4 ligation rejoins ends frequently introducing small insertions or deletions due to processing, causing frameshift-mediated knockout useful for disrupting negative regulators like MLO mildew susceptibility in barley. If homologous donor plasmid with homology arms flanking break provided in excess during S/G2 phase, Rad51 mediated homologous recombination uses donor as template copying desired edits resulting precise gene replacement or insertion. DSB stimulates HR frequency up to 1000-fold over spontaneous. Detection of editing uses Surveyor assay, T7E1 cleavage, deep amplicon sequencing showing indel signatures distinct for each repair outcome.

Ref: Jasin Rothstein CSH Perspect 2013 DSB repair; Carroll Genetics 2011 ZFN breaks DSB.

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Transgenic Plant -2

Zinc finger nucleases (ZFNs) consist of:

Zinc finger nucleases represent first generation programmable nucleases fusing custom DNA recognition to cleavage effector. Each ZFN monomer architecture includes tandem array of Cys2-His2 zinc finger motifs originally derived from transcription factor Zif268 and Sp1, each finger ~30 amino acids folded into beta-beta-alpha structure where Zn2+ tetrahedrally coordinated by two cysteines in beta hairpin and two histidines in alpha helix stabilizing domain. Recognition helix positions -1 to 6 contact major groove bases via hydrogen bonds and van der Waals contacts. Array of three to six fingers recognizes 9 to 18 base pairs contiguously. C-terminal FokI endonuclease domain from Flavobacterium okeanokoites comprises nonspecific cleavage domain 196 amino acids requiring dimerization for double strand break formation leaving 4 bp 5' overhangs. Flexible linker GSGGS between finger array and FokI permits optimal spacing. Pair of ZFNs binding opposite strands tail-to-tail spaced 5-7 bp allows FokI dimer formation cutting intervening sequence, creating DSB stimulating homologous recombination or mutagenic NHEJ for targeted knockout or knockin applications in crops and human therapeutics for CCR5 editing and hemophilia correction.

Ref: Kim et al. PNAS 1996 ZFN; Carroll Annu Rev Biochem 2014 ZFN; Chandrasegaran 2016.

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Gene Tar getting

Which genome editing platform uses protein-DNA interactions to determine specificity?

Genome editing specificity can derive from protein-DNA contacts versus RNA-DNA base pairing. CRISPR Cas9 relies on guide RNA pairing and PAM. RNA interference uses small RNA-mRNA complementarity. Zinc finger nucleases use engineered arrays of Cys2His2 zinc finger domains, each recognizing three nucleotides via alpha helix contacting major groove. Tandem fingers create specific protein-DNA interface fused to FokI nuclease. Thus ZFN specificity is entirely determined by protein side chain interactions with DNA bases, requiring modular assembly and selection to achieve desired target without RNA intermediate.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

Which genome editing method does not require dimerization?

Zinc finger nucleases and TALENs use FokI endonuclease domain which lacks catalytic activity as monomer and must dimerize to cleave DNA. Therefore they require paired binding sites in tail-to-tail orientation separated by spacer. Meganucleases act as single protein but still involve large protein-DNA recognition interface. CRISPR Cas9 avoids protein dimerization; a single Cas9 protein guided by single guide RNA recognizes target via RNA-DNA pairing and PAM, then introduces double strand break. This monomeric architecture simplifies multiplexing and delivery for genome editing.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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