Genetics, Functional Genomics, and Perturbation Screens
Finding Function at Scale
Functional genomics
Modern molecular biology uses pooled and arrayed perturbations, sequencing-based readouts, and statistical modeling to infer causal gene function across pathways and networks.
Perturbation approaches
Screen types
Positive-selection screen
- Enriches cells with advantageous perturbations
- Useful for resistance phenotypes
Negative-selection screen
- Depletes cells with essential gene loss
- Useful for viability dependencies
Interpretation of screens
A screen identifies statistical dependencies, but validation requires follow-up experiments that test mechanism, specificity, and context dependence.
What is a major advantage of CRISPRi over nuclease-mediated knockout for some studies?
CRISPRi modulates expression without generating double-strand breaks, reducing certain confounders.
Correct answer: It avoids DNA double-strand breaks
Why are hit lists from genome-wide screens not sufficient on their own?
Screening is hypothesis-generating rather than definitive.
Correct answer: They require validation because off-target effects, context specificity, and statistical noise can produce false positives or negatives
Validation strategies
| Strategy | Purpose |
|---|---|
| Independent guide RNAs | Rule out guide-specific artifacts |
| Rescue experiments | Demonstrate causality |
| Orthogonal perturbation | Confirm mechanism by another method |
| Time-course analysis | Capture dynamic phenotypes |
Which screen is best suited to identify genes required for cell survival under baseline conditions?
Loss of essential genes leads to depletion from the population.
Correct answer: Negative-selection screen
What is one limitation of pooled CRISPR screens?
Pooled formats are powerful but not universal.
Correct answer: They often rely on phenotypes compatible with sequencing-based pooled readouts and can miss subtle or spatially restricted effects