Genome Engineering: Cut Site, Repair Choice and Editing Outcome · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.
En anglais

Cas9 cuts, and repair shapes the allele

Science · Genetics & Evolution · ages 22-24
Name ______________________   Date ____________

  1. A coding sequence reads ATG CAA GGT. End joining deletes CAA, exactly three bases. What follows?

    • The reading frame shifts, so protein function must be lost
    • The reading frame is restored, so the protein is unchanged
    • The reading frame is preserved, but protein function may change

  2. After Cas9 makes a break, what shapes the repaired DNA sequence?

    • Repair pathways and any templates they use
    • Guide pairing and target PAM recognition
    • Marker expression and the DNA delivery method

  3. A delivery-marker signal alone proves that the intended target allele was edited correctly.

    Circle one:   True   False

  4. A target base near a Cas9 break is C. Researchers want T there by HDR. Their proposed donor matches both target flanks but still carries C. Which revision supplies the intended repair information?

    • Keep donor C and lengthen both matching flanks
    • Replace donor C with T and keep both matching flanks
    • Replace donor C with T and use unrelated flanking DNA

  5. The same Cas9 and guide yield mainly indels in resting mammalian cells and more donor-directed changes in cycling cells. Which repair difference could explain this?

    • HDR is more available in cycling cells
    • End joining uses donors in cycling cells
    • HDR uses guides as templates in cycling cells

  6. Two guides cut their intended sites similarly. Only one has a predicted close match elsewhere beside a compatible PAM, with PAM-distant mismatches. All else equal, which warrants more off-target concern?

    • The guide without a predicted off-target match
    • The guide with the PAM-bearing close match
    • Both guides, since intended cutting is similar

  7. Providing a matching donor guarantees the intended knock-in in every cell that receives Cas9.

    Circle one:   True   False

  8. No edits are detected at two possible off-target sites using a test that misses very rare events. What limits a genome-wide claim of zero off-target edits?

    • The tested sites establish that every genomic site is unchanged
    • The stated detection limit rules out all edits at those sites
    • Rare edits at tested sites or edits elsewhere could escape detection
LightMySky · lightmysky.comW1-mt_3DqvkO4wdE-s1

Answer key

For grown-ups. Fold this page away before handing over the rest.

Cas9 cuts, and repair shapes the allele W1-mt_3DqvkO4wdE-s1

  1. The reading frame is preserved, but protein function may change · The sequence becomes ATG GGT. Removing three bases preserves the reading frame, but deleting a codon may affect the protein.
  2. Repair pathways and any templates they use · Target recognition positions the break. Repair pathways and any template they use shape the repaired sequence.
  3. False · A marker signal does not read the target DNA. The intended allele needs a separate sequence check.
  4. Replace donor C with T and keep both matching flanks · The donor must carry T to supply the intended correction, while retaining matching flanks. Longer arms alone do not supply T, and this revision does not guarantee HDR.
  5. HDR is more available in cycling cells · HDR is often favoured in S and G2. Cycling cells may use a donor more often, while other repair pathways still compete.
  6. The guide with the PAM-bearing close match · The PAM-bearing close match is a possible unintended target. PAM-distant mismatches may be tolerated, so it warrants testing.
  7. False · A donor supplies sequence information, but competing repair can produce other outcomes. The intended allele must be checked.
  8. Rare edits at tested sites or edits elsewhere could escape detection · The result covers the two tested sites within the assay's detection limit. It cannot exclude rarer events or edits elsewhere.
Worksheet · LightMySky