Practice question

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.