Crispresso Editing

What the source does

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• NetworkNone observedNo outbound calls in the source
• ShellNone observedNo commands run in the source
• DependenciesPinnedNothing floats
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Five audit layers

Gates applied: no behavioural pass.

Audit 0.4.1 · source sha c8c1fc3a2b13 · 19 Oct 2026Report an issue or request a re-scan

• PublisherGPTomics GitHub organizationOwner of the repository this skill is read from
• Source folderGPTomics/bioSkills/crispr-screens/crispresso-editingThe exact folder we audited
• LicenceMITAs the repository states it
• Commit pinned for this verdictc8c1fc3a2b13A new audit runs when the source changes
• Repository statusArchivedLast push 65 days ago
• Sandboxed runNot runSkills are read, not executed; layer L4 is skipped

Its description: Quantifies CRISPR editing outcomes with CRISPResso2 (Clement 2019 Nat Biotechnol) across Cas9-nuclease (indels, HDR), CBE and ABE base editors (target conversion + bystander), and prime editor (pegRNA-templated) modes.…

---
name: bio-crispr-screens-crispresso-editing
description: Quantifies CRISPR editing outcomes with CRISPResso2 (Clement 2019 Nat Biotechnol) across Cas9-nuclease (indels, HDR), CBE and ABE base editors (target conversion + bystander), and prime editor (pegRNA-templated) modes. Covers single-amplicon (CRISPResso), multi-sample batch (CRISPRessoBatch), pooled-amplicon (CRISPRessoPooled), WGS off-target (CRISPRessoWGS), and sample-comparison (CRISPRessoCompare) workflows; quantification-window math that controls what is called edited; substitution-vs-indel diagnostic to distinguish BE from Cas9 contamination; MMEJ deletion pattern interpretation; allele-frequency tables; and failure modes from amplicon misalignment or contamination. Use when quantifying editing from amplicon sequencing, choosing CRISPResso mode by design, distinguishing intended edits from bystanders and indel byproducts, debugging low-alignment runs, or generating publication-grade editing reports.
tool_type: cli
primary_tool: CRISPResso2
---

## Version Compatibility

Reference examples tested with: CRISPResso2 2.2.14+ (pinellolab/CRISPResso2), pandas 2.2+, numpy 1.26+, matplotlib 3.8+.

Before using code patterns, verify installed versions match. If versions differ:
- CLI: `CRISPResso --version`; `CRISPRessoBatch --help`; `CRISPRessoPooled --help`; `CRISPRessoWGS --help`; `CRISPRessoCompare --help`
- Python: `from CRISPResso2 import ...`

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

## CRISPResso2 Editing Quantification

**"Quantify CRISPR editing from my amplicon sequencing"** -> Align amplicon reads against the reference, classify each read as unmodified / NHEJ / HDR / base-edited / prime-edited within the quantification window, and report per-edit-type frequencies, indel size distributions, allele-frequency tables, and substitution-position profiles.

- CLI: `CRISPResso` -- single amplicon, single sample
- CLI: `CRISPRessoBatch` -- multi-sample with per-sample parameters
- CLI: `CRISPRessoPooled` -- multi-amplicon pooled amplicon sequencing
- CLI: `CRISPRessoWGS` -- off-target quantification from whole-genome BAM
- CLI: `CRISPRessoCompare` -- pairwise outcome comparison (e.g., treated vs untreated)

## Mode Decision Tree

| Experimental design | Mode | Key parameters |
|---------------------|------|----------------|
| Single amplicon, single sample (e.g. pilot edit validation) | `CRISPResso` | `--amplicon_seq`, `--guide_seq` |
| Same amplicon, many samples (e.g. timecourse, dose response) | `CRISPRessoBatch` | `--batch_settings` table |
| Many amplicons, pooled in one library (e.g. arrayed validation pool) | `CRISPRessoPooled` | `--amplicons_file` |
| Off-target survey from whole-genome BAM | `CRISPRessoWGS` | `--bam_file`, `--reference_file`, `--region_file` |
| Comparing two CRISPResso runs (e.g. condition A vs B) | `CRISPRessoCompare` | two positional output folders |
| HDR / knock-in validation | `CRISPResso` with `--expected_hdr_amplicon_seq` | Same as base CRISPResso |
| Cytosine base editor (C->T) | `CRISPResso --base_editor_output` | `--conversion_nuc_from C --conversion_nuc_to T` |
| Adenine base editor (A->G) | `CRISPResso --base_editor_output` | `--conversion_nuc_from A --conversion_nuc_to G` |
| Prime editor (templated edit) | `CRISPResso` with pegRNA parameters | `--prime_editing_pegRNA_spacer_seq`, `--prime_editing_pegRNA_extension_seq`, `--prime_editing_pegRNA_scaffold_seq` |

**Fails when:**
- Pooled-amplicon mode applied to amplicons that share primer sequences -- reads get misassigned.
- Base editor mode without specifying `--conversion_nuc_from`/`--conversion_nuc_to` -- defaults assume CBE (C->T); ABE runs will misclassify.
- Prime editor mode without `--prime_editing_pegRNA_extension_seq` -- the RTT template is missing, no edit is detectable.

## The Quantification Window

**Why this matters for postdoc-level use:** CRISPResso classifies reads as "edited" or "unmodified" based on whether *modifications fall inside the quantification window* (not the whole amplicon). The window is centered on the predicted cut site (Cas9: 3 bp upstream of PAM; Cas12a: 18 bp downstream of PAM) with a default size of 1. `--quantification_window_size N` extends N bp on EACH side, so the window is 2N bp wide.

```bash
# Default Cas9 setup
--quantification_window_size 1                  # 1-bp window at cut site
--quantification_window_center -3               # 3 bp upstream of PAM

# Base editor: widen window to cover editing positions 4-8
--quantification_window_size 10                 # 10 bp each side (20 bp total)
--quantification_window_center -10              # center on the editing window
```

**Consequences of mis-sized window:**
- Too narrow: misses edits at HDR positions or far bystanders; underestimates editing
- Too wide: includes random sequencing errors; inflates editing rate
- Wrong center: edits at correct position are scored as outside the window

For base editing screens a widened window is conventional; CRISPResso2's own base-editor guidance uses `--quantification_window_center -17` with a window sized to span the editing positions. For prime editing with multi-base templated edits, widen to encompass the entire edit region.

## Single-Amplicon Cas9 Editing

**Goal:** Quantify indel frequencies and HDR efficiency from a single target site.

**Approach:** Align FASTQ reads to the reference and (optional) expected-HDR amplicon, classify each read, and report aggregated statistics.

```bash
CRISPResso \
    --fastq_r1 sample_R1.fastq.gz \
    --fastq_r2 sample_R2.fastq.gz \
    --amplicon_seq <amplicon_sequence_ref_genome> \
    --guide_seq <20nt_protospacer_no_PAM> \
    --expected_hdr_amplicon_seq <edited_amplicon_for_HDR> \  # OPTIONAL
    --quantification_window_size 1 \
    --quantification_window_center -3 \
    --min_average_read_quality 30 \                          # Phred quality

Excerpt, first 6,000 characters of SKILL.md, verbatim from the audited commit. It is text from someone else’s repository, quoted so you can judge it before your agent reads it.

pip install pandas matplotlib seaborn

git clone https://github.com/GPTomics/bioSkills
git -C bioSkills checkout c8c1fc3a2b13

Works in

1. 2026-10-19
   ✓SAFE B 89 / 100

   first audit · source c8c1fc3a2b13

Questions about Crispresso Editing

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