Perturb seq analysis

Version Compatibility

Reference examples tested with: Pertpy 0.6+, SCEPTRE 0.10+ (R / katsevich-lab/sceptre), Mixscape via Seurat 4.3+ or Pertpy, scanpy 1.10+, anndata 0.10+, pandas 2.2+, numpy 1.26+, scipy 1.12+.

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show pertpy scanpy anndata
• R: packageVersion('sceptre'); ?sceptre; ?Seurat::PrepLDA

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

Single-Cell Perturb-Seq Analysis

"Analyze a single-cell pooled CRISPR perturbation screen" -> Assign sgRNAs to cells, filter unperturbed escapers, normalize counts, fit per-gene differential expression conditioned on perturbation, and rank perturbations by their molecular effect.

• Python: pertpy unified framework for Mixscape + SCEPTRE-via-R + differential expression
• R: sceptre for low-MOI NB GLM + permutation testing
• Python/R: Seurat::MixscapeLDA and downstream

Experimental Architecture Comparison

Method	Year	Architecture	Readout	MOI	Single-cell sgRNA detection
Perturb-seq (Dixit 2016, Cell)	2016	sgRNA expressed in cassette; direct PCR capture	scRNA-seq	High (4-50 sgRNAs/cell)	Yes via amplicon-PCR pre-sequencing
CROP-seq (Datlinger 2017, Nat Methods)	2017	sgRNA expressed via tRNA-spacer-sgRNA-pgk-puro; barcoded in 3'UTR of fluorescent marker; captured in 3' droplet	scRNA-seq	Low (1-2 sgRNAs/cell)	Native via 10X 3' chemistry
Perturb-CITE-seq (Frangieh 2021, Nat Genet)	2021	Adds surface-protein hashtag oligos to CROP-seq	scRNA-seq + ADT (protein)	Low	CROP-seq architecture
ECCITE-seq (Mimitou 2019, Nat Methods)	2019	Surface-protein hashtag with sgRNA-marked cells	scRNA-seq + ADT	Low	Hash + sgRNA
Perturb-ATAC (Rubin 2019, Cell)	2019	scATAC-seq readout	scATAC	Low	sgRNA capture via separate library prep
Perturb-multiome (10X)	2021+	scRNA + scATAC simultaneously	scRNA + ATAC	Low	Direct capture from sgRNA cassette
Replogle GW Perturb-seq (2022, Cell)	2022	Multiplexed CRISPRi with sgRNA barcoding	scRNA-seq	1 sgRNA/cell	Direct capture

Decision rule: For genome-wide CRISPRi screens, Replogle's CRISPRi + 10X 3' direct-capture protocol is the gold standard (2.5M cells across 2,058 perturbations in Replogle 2022). For protein readout, Perturb-CITE-seq. For chromatin, Perturb-multiome. For low-throughput pilot, original Dixit Perturb-seq.

MOI and sgRNA Assignment

The central technical challenge: Each cell must receive exactly one sgRNA (otherwise the perturbation is undefined). At MOI 0.3, ~26% of cells get ≥1 sgRNA, but 4% get ≥2; the cells with multiple sgRNAs must be filtered or analyzed as combinatorial perturbations.

Assignment workflow:

1. Detect sgRNA reads per cell: From the sgRNA library prep (direct capture or 3'UTR barcode), count reads per sgRNA per cell.
2. Threshold: Most pipelines use 10+ reads of one sgRNA to assign that perturbation.
3. Multiplets: Cells with 2+ sgRNAs at >10 reads each are either multi-perturbed (analyzable as combinatorial) or doublets.
4. Doublet detection: Use scDblFinder, Scrublet, or AMULET (multiome) to identify doublets independently from sgRNA assignment.

Goal: Assign a single perturbation identity (or 'multiplet'/'none') to every cell from the sgRNA counts matrix.

Approach: Threshold per-cell sgRNA reads at ≥10 (Pertpy convention); cells exceeding the threshold for exactly one sgRNA are assigned that perturbation; cells with multiple sgRNAs above threshold are flagged as multiplets for filtering or combinatorial analysis.

# sgRNA assignment via threshold counting
def assign_sgrna(adata, sgrna_counts_layer='sgrna_counts', threshold=10):
    '''Per-cell sgRNA assignment. Returns single assignment or 'multiplet'/'none'.'''
    import numpy as np
    counts = adata.layers[sgrna_counts_layer]  # cells x sgRNAs
    above_thresh = counts >= threshold
    n_sgrna_per_cell = above_thresh.sum(axis=1)
    assignments = np.where(
        n_sgrna_per_cell == 0, 'none',
        np.where(n_sgrna_per_cell == 1,
                  [adata.var_names[i] for i in counts.argmax(axis=1)],
                  'multiplet'))
    adata.obs['sgrna_assignment'] = assignments
    return adata

Escaper Cell Filtering (Mixscape)

Why this matters: Not all sgRNA-positive cells actually edit. 30-60% of cells may receive sgRNA but fail to undergo gene knockdown ("escapers"). Including escapers dilutes the perturbation effect; Mixscape (Papalexi 2021) identifies and filters them.

Mixscape algorithm: For each perturbed cell, compute a "perturbation signature" = (its expression) - (mean of K nearest non-targeting-control cells). This signature isolates the perturbation effect from cell-state variation. Cells with perturbation signature similar to NTC distribution are escapers.

import pertpy as pt
import scanpy as sc

# adata is a scRNA-seq AnnData with 'sgrna_assignment' column
# Pertpy 0.6+ Mixscape API (verify against installed pertpy with help(pt.tl.Mixscape))
mixscape = pt.tl.Mixscape()
mixscape.perturbation_signature(
    adata=adata,
    pert_key='sgrna_assignment',     # .obs column with sgRNA target per cell
    control='NTC',                   # name of non-targeting control in pert_key column
    n_neighbors=20,                  # K neighbors for KNN-NTC subtraction
)
# Writes .layers['X_pert'] with perturbation-signature-corrected expression

# Filter escapers: classify perturbed cells as KO (true perturbation) or NP (non-perturbed/escaper)
mixscape.mixscape(
    adata=adata,
    pert_key='sgrna_assignment',     # pert_key (not 'labels' in modern pertpy)
    control='NTC',
    new_class_name='mixscape_class', # .obs column to write
)
# Defaults to layer='X_pert' (output of perturbation_signature)

# Keep only KO cells for downstream analysis
adata_ko = adata[adata.obs['mixscape_class'].isin(['KO'])].copy()
print(f'KO cells: {adata_ko.n_obs} ({adata_ko.n_obs/adata.n_obs:.1%} of perturbed)')

Critical: Mixscape can fail when the perturbation has weak phenotype; empirically Mixscape detects perturbations with log-fold-change <-0.5 (depletion) reliably, but weaker effects collapse into the NTC distribution. For genome-wide screens, run Mixscape per perturbation; for low-effect perturbations, trust the assignment without filtering.

SCEPTRE for Low-MOI Differential Expression

Why this matters: Standard differential-expression tools (DESeq2, MAST) assume Gaussian-mixture distribution and fail at single-cell scale with sparse, zero-inflated data. SCEPTRE (Katsevich Lab, 2021; low-MOI variant Barry 2024 Genome Biol) uses a negative-binomial GLM with conditional resampling:

1. Per gene, fit NB GLM: log(expr_g) ~ pert_indicator + technical_factors
2. Compute z-score for the perturbation coefficient
3. Resample the pert_indicator (conditional on counts) 500-1000 times; compute permutation null
4. Get FDR via permutation; not parametric

library(sceptre)

# Input: sce object or sparse matrix + metadata
# Required: gene_expression_matrix, perturbation_indicator (binary per cell per pert),
#           technical_factors (batch, n_genes, etc.)

# For each gene + perturbation pair:
results <- run_sceptre_low_moi(
    expression_matrix    = gene_expr,            # genes x cells
    grouping_var         = pert_indicator,       # length(cells); 0 = NTC, 1 = perturbed
    technical_factors    = covariates_df,        # n_genes, n_umi, batch
    response_grouping_var = 'gene_id',
    n_permutations       = 1000
)
# Output: per-gene-per-pert p-value, log-fold-change, FDR

Advantage over MAST: SCEPTRE's permutation NB GLM is the only method that maintains calibrated FDR in pooled-screen scRNA-seq (Barry 2024 benchmark). MAST and Wilcoxon are over-confident due to data sparsity.

Pertpy Unified Framework

Pertpy (https://pertpy.readthedocs.io) integrates Mixscape, distance-based perturbation comparison, EdgeR/PyDESeq2/WilcoxonTest DE, and factor models in a single AnnData-based interface. For SCEPTRE specifically, invoke the R sceptre package separately (Pertpy does not wrap it).

import pertpy as pt
import scanpy as sc

# Load data
adata = pt.dt.papalexi_2021()  # built-in example from Mixscape paper

# Standard scRNA-seq preprocessing (scanpy)
sc.pp.normalize_total(adata, target_sum=1e4)
sc.pp.log1p(adata)
sc.pp.highly_variable_genes(adata, n_top_genes=2000)

# Mixscape escaper filtering (writes .layers['X_pert'] and .obs['mixscape_class'])
ms = pt.tl.Mixscape()
ms.perturbation_signature(adata, pert_key='perturbation', control='NT', n_neighbors=20)
ms.mixscape(adata, pert_key='perturbation', control='NT')

# Filter to KO cells
adata_ko = adata[adata.obs['mixscape_class'].isin(['KO', 'NT'])].copy()

# Pseudobulk differential expression via pertpy (PyDESeq2 backend)
de = pt.tl.PyDESeq2(adata_ko, design='~perturbation')
de.fit()
results_df = de.test_contrasts(contrast=('perturbation', 'GENE_X', 'NT'))

# For calibrated SCEPTRE on low-MOI single-cell data, use R sceptre directly
# (Barry 2024 Genome Biol; not bundled in pertpy)

Genome-Wide Perturb-Seq (Replogle 2022)

Replogle 2022 Cell 185:2559 demonstrated genome-wide Perturb-seq:

• 2.5M cells across 2,058 perturbations (essentially every expressed protein-coding gene)
• CRISPRi via dCas9-KRAB
• Native 10X 3' direct-capture for sgRNA
• ~1,000 cells per perturbation (enough for per-perturbation DE)
• Cluster-based analysis of perturbed cells reveals gene-program organization

Scaling principles:

• Cells per perturbation: 500-1,000 minimum for stable DE
• 10X channels: 10-30 channels at 5,000-10,000 cells each
• Cost: ~$50-100K for genome-scale

# Replogle-style genome-wide design
# Each cell -> 1 sgRNA (low MOI)
# Each gene -> 5 sgRNAs (CRISPRi)
# Each pert -> 1000 cells
# Total: 19,000 genes x 5 sgRNAs = 95,000 sgRNAs
# Cells needed: 2 million (covering all 19k genes at ~100 cells/gene/sgRNA)

Factor-Based Analysis

For complex perturbation responses, decompose the per-cell perturbation effect into shared latent factors:

import pertpy as pt

# FR-Perturb (Factor-Regularized Perturb-seq) decomposes perturbations into shared factors
fr = pt.tl.FRPerturb()
factors = fr.fit(adata, pert_key='perturbation', n_factors=20)
# Output: per-perturbation factor loadings; similar perturbations share factors

Multiomic Perturb-seq (RNA + ATAC)

For chromatin readout: Use 10X Multiome with CRISPRi/a; sgRNA assignment via the same scATAC-seq library.

# Multiome: scRNA + scATAC + sgRNA
# Use ArchR or Signac for ATAC integration
# Use Pertpy for RNA-side DE
import muon as mu
mdata = mu.MuData({'rna': adata_rna, 'atac': adata_atac})
# Joint differential analysis across modalities

Failure Modes

Low sgRNA detection per cell

Trigger: Direct-capture method on CROP-seq library, or 3'UTR barcoding on direct-capture library. Mechanism: Architecture mismatch -- the sgRNA can't be detected by the wrong library prep. Symptom: sgRNA assignment rate <50% of cells. Fix: Match library prep to architecture; for CROP-seq, use 10X 3' chemistry; for direct-capture Perturb-seq, use the Dixit amplicon-PCR pre-sequencing.

Mixscape filters too many cells as escapers

Trigger: Weak perturbation phenotype; Mixscape's NTC-subtracted signature is similar to NTC null. Mechanism: Mixscape assumes a detectable signal; weak knockdown is misclassified as escaper. Symptom: >50% of perturbed cells classified as "NP" (non-perturbed); known essentials show no effect. Fix: Lower Mixscape stringency; skip Mixscape for low-effect perturbations; verify Cas9 expression first.

Doublet contamination drives apparent multi-perturbation cells

Trigger: High cell density loading on 10X channels. Mechanism: Two cells in one droplet appear to carry two sgRNAs. Symptom: "Multiplet" rate >5% after sgRNA assignment. Fix: Reduce cell loading per channel (5,000-7,000 instead of 10,000); run Scrublet or scDblFinder; remove doublets before sgRNA assignment.

MAST or Wilcoxon over-call hits

Trigger: Using parametric DE tools on sparse, zero-inflated scRNA-seq. Mechanism: These tools assume Gaussian or simpler null; single-cell data has zero-inflation that makes them over-confident. Symptom: Thousands of significant DE genes per perturbation; FDR uncalibrated. Fix: Use SCEPTRE (permutation-based NB GLM); Barry 2024 benchmark shows this is the only method with calibrated FDR.

Genome-scale Perturb-seq with insufficient cells per perturbation

Trigger: <500 cells per perturbation in genome-scale experiment. Mechanism: DE estimation requires sufficient cells per condition; <500 lacks power for moderate effects. Symptom: Inconsistent hit calls across replicates; pathway analysis non-specific. Fix: Scale up cell numbers; or run focused (sub-genome) Perturb-seq with more cells per pert.

Quantitative Thresholds

Threshold	Value	Source / Rationale
MOI for single sgRNA per cell	0.3	Poisson math; ~26% infected, 4% multi-infected
sgRNA assignment threshold	≥10 reads of one sgRNA	Pertpy / direct-capture convention
Multiplet rate (post-doublet filter)	<5%	Typical 10X 3' chemistry
Mixscape escaper-filter (true KO retention)	30-60% of perturbed cells	Papalexi 2021
Cells per perturbation (DE power)	500-1,000 minimum	Replogle 2022
SCEPTRE permutations	1,000+	Barry 2024
Genes per cell (QC)	≥500-1,000	Standard scRNA QC
Mt% threshold	<15-20%	Standard scRNA QC
Doublet detection threshold	scDblFinder, Scrublet defaults	Methods agree

Common Errors

Error / symptom	Cause	Solution
Low sgRNA detection	Architecture mismatch	Match library prep
Too many escapers in Mixscape	Weak phenotype	Skip Mixscape; verify Cas9
Inflated DE hits	MAST / Wilcoxon used	Switch to SCEPTRE
Inconsistent gene effects between channels	Channel batch effect	Add channel as covariate in SCEPTRE
Multiplet rate >10%	Over-loading cells	Reduce loading; doublet filter
Per-pert DE with <100 cells	Insufficient power	Increase cell numbers; or accept low resolution

References

• Dixit A et al. 2016. Cell 167:1853. Original Perturb-seq.
• Datlinger P et al. 2017. Nat Methods 14:297. CROP-seq.
• Frangieh CJ et al. 2021. Nat Genet 53:332. Perturb-CITE-seq.
• Mimitou EP et al. 2019. Nat Methods 16:409. ECCITE-seq.
• Rubin AJ et al. 2019. Cell 176:361. Perturb-ATAC.
• Papalexi E et al. 2021. Nat Genet 53:322. Mixscape.
• Barry T, Mason K, Roeder K, Katsevich E. 2024. Genome Biol 25:124. SCEPTRE for low-MOI Perturb-seq.
• Replogle JM et al. 2022. Cell 185:2559. Genome-wide Perturb-seq.
• Heumos L et al. 2023. Nat Methods 20:1349. Pertpy framework.
• Cao J et al. 2023. Nat Genet 55:1894. Mixscale (dose-response Perturb-seq).

Related Skills

• crispr-screens/library-design - Direct-capture vs CROP-seq library design
• crispr-screens/screen-qc - sgRNA assignment rates as QC
• crispr-screens/mageck-analysis - Pseudobulk analysis as alternative
• crispr-screens/hit-calling - Pseudo-bulk hit calling alternative
• single-cell/preprocessing - scRNA-seq preprocessing
• single-cell/clustering - Post-DE clustering
• single-cell/multimodal-integration - Multiome Perturb-seq
• single-cell/perturb-seq - General single-cell screen analysis
• pathway-analysis/go-enrichment - Pathway enrichment of perturbation hits