Pooled Genetic Screens: Library Design, Selection and Reading a Hit List · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.

One pool, thousands of guides

Science · Genetics & Evolution · ages 23-24
Name ______________________   Date ____________

  1. Cell count equals library size times coverage. A 20,000 guide library at 500 fold coverage needs how many cells? Type the number.

    Answer: ______________

  2. What is the basic design of a pooled screen?

    • One pool carries a guide library with one guide per cell, then selection keeps wanted cells
    • Each gene gets its own separate flask and technician
    • Cells are selected first and guides are added afterward

  3. You want the list of genes a cell cannot live without. Which selection design fits?

    • Positive selection, keeping the survivors
    • No selection at all
    • Negative selection, finding the dropouts

  4. On your hit list one guide scores strongly while three guides against the same gene stay silent. What do you conclude?

    • The gene is your best hit
    • The lone guide is suspect, likely a one guide artefact
    • The silent guides prove the gene is essential

  5. You want cells that survive a new drug. Which selection design fits?

    • Negative selection, finding the dropouts
    • Positive selection, keeping the survivors
    • A screen with no selection step

  6. What are non targeting guides for?

    • Measuring background noise in the assay
    • Cutting every gene at once
    • Replacing the selection step

  7. Your screen finishes and even the guides for known essentials show no depletion. What do you conclude?

    • No gene is essential in this cell type
    • The assay itself failed, since its built in check did not respond
    • Negative selection is always silent

  8. An 8,000 guide library needs 500 fold coverage, where cell count equals library size times coverage. Type the cells needed.

    Answer: ______________

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Answer key

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

One pool, thousands of guides W1-mt_0n-a7GfLOL-s1

  1. 10000000 · 20,000 times 500 is 10,000,000.
  2. One pool carries a guide library with one guide per cell, then selection keeps wanted cells · One pool, one guide per cell, then selection turns biology into counting.
  3. Negative selection, finding the dropouts · Essential genes reveal themselves by disappearing, which is negative selection.
  4. The lone guide is suspect, likely a one guide artefact · Agreeing guides rule out artefacts, so a lone hit with silent partners should not be trusted.
  5. Positive selection, keeping the survivors · Drug resistance leaves living winners in your hands, which is positive selection.
  6. Measuring background noise in the assay · Non targeting guides set the baseline, so real enrichment stands out from noise.
  7. The assay itself failed, since its built in check did not respond · Known essentials are the positive control: if they stay silent, the assay did not work.
  8. 4000000 · 8,000 times 500 is 4,000,000.
Worksheet · LightMySky