Crispr Screens Drugz Chemogenomic

---
name: crispr-screens-drugz-chemogenomic
description: Analyzes CRISPR drug-modifier (chemogenomic) screens with drugZ (Li & Hart 2019 Genome Med), a bidirectional Z-score method that identifies synthetic-lethal sensitizing genes and resistance-conferring suppressor genes from vehicle vs drug comparisons. Covers vehicle-anchored design (not Day-0), the bidirectional Z math giving 2-3x sensitivity over MAGeCK / STARS / edgeR / RIGER on drug screens, per-gene sumZ and normZ, synth (sensitizer) vs supp (suppressor) FDR, multi-dose handling, integration with control sgRNAs, and comparison with MAGeCK MLE with dose covariate. Use when running a drug-modifier CRISPR screen, identifying sensitizing or resistance genes for a drug candidate, choosing drugZ vs MAGeCK MLE for chemogenomic analysis, troubleshooting low-effect drug screens where MAGeCK lacks sensitivity, or designing a drug-screen layout (vehicle vs drug arms).
tool_type: cli
primary_tool: drugZ
---

## Version Compatibility

Reference examples tested with: drugZ Aug-2019+ (hart-lab/drugz; Python 3.6+), MAGeCK 0.5.9+, pandas 2.2+, numpy 1.26+, scipy 1.12+, statsmodels 0.14+, matplotlib 3.8+.

Before using code patterns, verify installed versions match. If versions differ:
- CLI: `drugz --version`; `python drugz.py --help`
- GitHub: install via `git clone https://github.com/hart-lab/drugz`

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

## drugZ Chemogenomic Analysis

**"Identify genes that sensitize or confer resistance to my drug in a CRISPR screen"** -> Compare drug-treated vs vehicle-treated arms (NOT Day-0 baseline) using bidirectional Z-scores per sgRNA, sum to per-gene normalized Z, and rank genes for sensitizer (synthetic lethal) vs suppressor (resistance) phenotype.

- CLI: `python drugz.py -i counts.txt -o drugz.txt -c Vehicle_r1,Vehicle_r2 -x Drug_r1,Drug_r2`
- Python: programmatic via `drugz.drugz_analysis()` (internal Python module)
- Workflow: vehicle-anchored counts -> Z-scoring -> per-gene summation -> direction-specific FDR

## Why drugZ for Drug Screens (not MAGeCK)

| Property | drugZ | MAGeCK RRA | MAGeCK MLE |
|----------|-------|------------|-------------|
| Bidirectional sensitivity | YES (sensitizer + resistance same scale) | Asymmetric (neg/pos separately) | Asymmetric |
| Drug-anchored baseline | YES (drug vs vehicle) | Either (drug vs vehicle or vs Day 0) | Either |
| Sensitivity to small effects | 2-3x higher (Li & Hart 2019 benchmark) | Lower | Lower |
| Statistical framework | Z-score from per-sgRNA NB residuals | NB + alpha-RRA | NB GLM with design matrix |
| Handles guide-level noise | sgRNA-level z aggregation | Rank-based aggregation | Built-in guide-efficacy term (optional) |
| Best for | Drug-modifier / chemogenomic screens | General essentiality / standard 2-condition | Time course / multi-condition |

**Why MAGeCK is suboptimal for drug screens:** MAGeCK's RRA was designed for two-condition essentiality; drug-vs-vehicle screens often have small effect sizes (10-30% sgRNA shift) that RRA rank-based aggregation under-detects. drugZ uses parametric Z-scoring tuned for these small effects.

**Quantified gain (Li & Hart 2019):** On DNA damage response chemogenomic screens, drugZ identified 2-3x more hits than STARS, MAGeCK, edgeR, or RIGER at the same FDR threshold; the additional hits were enriched in the expected pathway (DDR).

## The drugZ Algorithm (under the hood)

1. For each sgRNA, compute log2-fold-change drug vs vehicle: `LFC_drug_vs_veh`
2. Compute the empirical Z-score from a fitted Gaussian over the bulk distribution: `Z = (LFC - median(LFC)) / MAD(LFC)`
3. Per gene, sum Z across all sgRNAs targeting it: `sumZ = sum(Z_sgRNA)`
4. Normalize for the number of sgRNAs: `normZ = sumZ / sqrt(N_sgrna)`
5. Compute two-sided p-value per gene: synth (sensitizer = negative normZ) and supp (resistance = positive normZ)
6. Benjamini-Hochberg FDR correction per direction

**Critical:** Vehicle vs drug, NOT Day 0 vs drug. Day-0 baseline conflates proliferation effects with drug effects.

## Run drugZ on a Drug-Modifier Screen

**Goal:** Quantify per-gene sensitizing and suppressor effects from a chemogenomic screen.

**Approach:** Run `drugz.py` with vehicle and drug sample columns; output per-gene sumZ, normZ, and direction-specific p-values + FDR.

```bash
git clone https://github.com/hart-lab/drugz
cd drugz

# Standard drug screen comparison:
# Vehicle (DMSO or carrier) replicates: Veh_r1, Veh_r2, Veh_r3
# Drug-treated replicates: Drug_r1, Drug_r2, Drug_r3

python drugz.py \
    -i counts.txt \                       # input read-count file (tab-separated)
    -o drugz_output.txt \                  # output file
    -c Veh_r1,Veh_r2,Veh_r3 \              # control samples (comma-separated)
    -x Drug_r1,Drug_r2,Drug_r3 \           # treated samples (comma-separated)
    -r control_genes.txt \                 # OPTIONAL: genes to exclude
    -p 5                                   # pseudocount (default 5)

# Output: drugz_output.txt with columns:
#   GENE, numObs, sumZ, normZ, pval_synth, rank_synth, fdr_synth, pval_supp, rank_supp, fdr_supp
```

**Output columns:**

| Column | Meaning |
|--------|---------|
| `GENE` | Gene symbol |
| `numObs` | Number of sgRNAs contributing |
| `sumZ` | Summed per-sgRNA Z-score |
| `normZ` | Normalized Z = sumZ / sqrt(N) |
| `pval_synth` | One-sided p-value for sensitizer (negative effect; gene KO sensitizes to drug) |
| `rank_synth` | Rank for sensitizers |
| `fdr_synth` | BH-corrected FDR for sensitizers |
| `pval_supp` | One-sided p-value for suppressor (positive effect; gene KO confers resistance) |
| `rank_supp` | Rank for suppressors |
| `fdr_supp` | BH-corrected FDR for suppressors |

**Interpretation:**
- Sensitizers (synthetic lethal): `fdr_synth < 0.05` -- loss of these genes makes cells more sensitive to drug. Examples: PARPi targets BRCA1/2; cisplatin sensitizes ERCC.
- Suppressors (resistance): `fdr_supp < 0.05` -- loss of these genes confers resistance. Examples: drug-efflux genes; drug target itself paradoxically.

## Vehicle vs Day-0 Reference: Critical Decision

**Why this matters:** Drug screen analysis can compare drug to:
1. **Vehicle (DMSO / carrier)** -- isolates drug-specific effect; correct anchor.
2. **Day 0 (initial library)** -- conflates proliferation, drug, and vehicle effects.

```
counts at Day 0          (no perturbation; cloning baseline)
    |
    v
counts at Day 7 - Vehicle (proliferation only; what survives in normal culture)
counts at Day 7 - Drug    (proliferation + drug effect)
    |
    v
Drug effect = LFC(Drug vs Vehicle)         # CORRECT
Wrong:       LFC(Drug vs Day 0)            # confounds drug with general proliferation
```

drugZ specifically requires `--control-samples` to be vehicle samples. Always include matched vehicle controls in drug screens.

## Drug-Dose and Time-Course Designs

**drugZ for dose-response:** Not natively designed for dose; instead, run drugZ separately at each dose vs vehicle, then look for genes with consistent direction across doses.

```bash
for DOSE in low mid high; do
    python drugz.py \
        -i counts.txt \
        -o drugz_${DOSE}.txt \
        -c Veh_r1,Veh_r2 \
        -x Drug${DOSE}_r1,Drug${DOSE}_r2
done

# Then aggregate: genes significant at high dose AND consistent direction at mid/low dose
```

**For multi-condition drug-screens** (time × drug × cell-line), use MAGeCK MLE with explicit design matrix instead -- MLE handles multi-factorial; drugZ does not.

## Comparison: drugZ vs MAGeCK MLE for Drug Screen

**Goal:** When to use each method.

| Question | drugZ | MAGeCK MLE |
|----------|-------|-------------|
| Single drug, single dose, vehicle vs drug | YES (preferred) | Acceptable |
| Multiple doses, drug response curve | Per-dose drugZ + meta | YES (preferred with dose covariate) |
| Time course at single dose | Per-timepoint drugZ + meta | YES (preferred with time covariate) |
| Drug + cell-line panel | Per-line drugZ + meta | YES (or Chronos) |
| Combinatorial drug pairs | Per-pair drugZ + meta | YES (preferred with interaction) |
| Synergy / antagonism detection | Limited (per-drug calling only) | YES (interaction term in MLE) |
| Small effect sizes (LFC <0.5) | Highest sensitivity | Lower sensitivity |
| Heavy selection (>40% guides change) | OK | Norm needs control sgRNAs |

**Reconciliation:** For simple drug-modifier screens with one drug and one vehicle, run both drugZ and MAGeCK MLE; hits called by both are high confidence; drugZ-only hits at low LFC need orthogonal validation (drug + arrayed validation).

## Removing Genes from Null Distribution

**Goal:** Exclude reference essential or control genes from the Z-score null distribution.

**Approach:** Provide `-r` with a file listing gene symbols whose sgRNA-level Z scores should not influence the null. Useful when CEGv2 essentials would otherwise inflate the null distribution.

```bash
# Pass a file with one gene per line
cat > remove_essential.txt <<EOF
RPS3
RPL11
EIF3A
POLR2A
CDK1
EOF

python drugz.py \
    -i counts.txt \
    -o drugz_clean.txt \
    -c Veh_r1,Veh_r2 \
    -x Drug_r1,Drug_r2 \
    -r remove_essential.txt
```

**When to use:** If pilot drugZ runs show many essential genes appearing as "sensitizers" purely because they drop out under any condition, removing them gives a cleaner drug-specific signal.

## Failure Modes

### drugZ shows no synthetic-lethal hits despite known sensitizing genes

**Trigger:** Comparing drug vs Day-0 instead of drug vs vehicle.
**Mechanism:** Day-0 comparison conflates drug effect with normal-culture proliferation; essential genes drop in both conditions, masking drug-specific sensitization.
**Symptom:** PARPi screen shows no sensitization at BRCA1/BRCA2 despite expected biology.
**Fix:** Re-run with vehicle samples as `--control-samples`. The drug-vs-vehicle is the canonical comparison.

### High false-positive rate among essential genes

**Trigger:** Essential genes drop out in both vehicle and drug arms; small relative shift gives misleadingly high Z.
**Mechanism:** drugZ's Z-score is symmetric; essential genes drop in both arms but slightly more in drug -> "synthetic lethal" call.
**Symptom:** Hit list dominated by RPS, RPL, EIF essentials.
**Fix:** Use `--remove-genes-file` to exclude CEGv2 essentials; or filter the output post-hoc.

### Inconsistent results between repeats of drugZ

**Trigger:** Insufficient sgRNAs per gene; small effect sizes.
**Mechanism:** drugZ's per-gene sumZ depends on enough sgRNAs to be stable; with 3-4 sgRNAs/gene, single-guide noise drives variation.
**Symptom:** Same data produces different top hits across repeated runs.
**Fix:** Use a 6+ sgRNAs/gene library (Avana, Dolcetto); or aggregate multiple drugZ runs with different bootstrap seeds; or use MAGeCK MLE for stability.

### drugZ ignores dose information

**Trigger:** Multi-dose screen analyzed at highest dose only.
**Mechanism:** drugZ doesn't model dose; running at one dose loses the dose-response information.
**Symptom:** Hits at high dose may be dose-specific (not true responders).
**Fix:** Run drugZ at each dose; require consistency across doses for high-confidence hits.

### Drug-target gene appears as "suppressor"

**Trigger:** Loss of drug target reduces drug binding, increasing drug resistance.
**Mechanism:** Real biology -- drug target itself is a resistance gene from a KO perspective.
**Symptom:** Drug-target gene like PARP1 appears in suppressor list for PARPi screen.
**Fix:** Expected biology. Annotate the drug target separately. The suppressor list is correct.

## Quantitative Thresholds

| Threshold | Value | Source / Rationale |
|-----------|-------|--------------------|
| Sensitizer hit | `fdr_synth < 0.05` | Li & Hart 2019; BH-corrected |
| Suppressor hit | `fdr_supp < 0.05` | Same |
| High-confidence sensitizer | `fdr_synth < 0.01 AND normZ < -3` | Conservative |
| Pseudocount default | 5 | Li & Hart 2019 |
| Min sgRNAs per gene for stable Z | 4-6 | Below this, Z varies between runs |
| Vehicle replicates needed | 3+ | For stable Z null distribution |
| Drug replicates needed | 3+ | For per-gene sumZ stability |

## Common Errors

| Error / symptom | Cause | Solution |
|-----------------|-------|----------|
| No hits | Wrong control samples (Day-0 instead of vehicle) | Re-run with vehicle |
| Hits dominated by essentials | Essentials inflate null | Use `--remove-genes-file` with CEGv2 |
| Unstable hits across runs | Too few sgRNAs/gene | Use 6+ sgRNAs/gene library |
| Drug-target appears in suppressor | Real biology | Annotate separately |
| MAGeCK and drugZ disagree | Different statistical sensitivity | drugZ more sensitive; trust for chemogenomic |
| Inconsistent between doses | Real dose effect | Require consistency across doses |

## References

- Li G & Hart T. 2019. *Genome Medicine* 11:52. drugZ algorithm and benchmark.
- Aregger M et al. 2020. *Mol Cell* 80:577. Original chemogenomic screens with TKOv3 library.
- Olivieri M et al. 2020. *Cell* 182:481. DDR chemogenomic screens with drugZ.
- Behan FM et al. 2019. *Nature* 568:511. Sanger Score; drug-modifier panels.
- Pacini C et al. 2021. *Cell Syst* 12:1132. Benchmark of drug-modifier methods.

## Related Skills

- crispr-screens/mageck-analysis - MAGeCK MLE alternative for multi-condition drug screens
- crispr-screens/bagel-essentiality - BAGEL2 alternative; sensitive to tumor-suppressor / drug-target
- crispr-screens/hit-calling - Cross-method decision tree including drugZ
- crispr-screens/screen-qc - Pre-drugZ QC including replicate concordance
- crispr-screens/library-design - 6+ sgRNAs/gene library for stable Z
- crispr-screens/copy-number-correction - Pre-correction for cancer-line drug screens
- crispr-screens/base-editing-analysis - Variant-function drug-modifier screens
- pathway-analysis/go-enrichment - Functional analysis of drug-modifier hits
- clinical-databases/clinvar-lookup - Clinical interpretation of drug targets