CRISPRDesigner2

A CRISPR gRNA design tool for SpCas9 (NGG PAM) targeting hg38 and mm10 genomes. Available as both a Python package for programmatic use and a Snakemake workflow for batch processing.

Overview

This tool designs CRISPR guide RNAs (gRNAs) for SpCas9 targeting NGG PAM sites:

1. Extracts candidate gRNAs from input genomic regions
2. Filters guides based on sequence quality criteria
3. Scores on-target efficiency using the RS3 model
4. Scores off-target specificity using GuideScan2
5. Outputs scored guides in standard formats

Installation

Option 1: Python package (recommended for integration)

Install as an editable package for use in other projects:

# Clone the repository
git clone https://github.com/EngreitzLab/CRISPRDesigner2.git
cd CRISPRDesigner2

# Create conda environment
conda env create -f workflow/envs/CRISPRDesigner2.yaml -n CRISPRDesigner2
conda activate CRISPRDesigner2

# Install as Python package
pip install -e .

Option 2: Snakemake workflow only

If you only need the Snakemake workflow:

conda env create -f workflow/envs/CRISPRDesigner2.yaml -n CRISPRDesigner2
conda activate CRISPRDesigner2

Data downloads

Genome FASTA

# hg38
wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
gunzip hg38.fa.gz

# mm10
wget https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/mm10.fa.gz
gunzip mm10.fa.gz

GuideScan2 index

Download pre-built indices from guidescan.com/downloads, then create a BAM index:

module load biology samtools/1.16.1
samtools index path/to/guidescan2.bam.sorted

RS3 model weights

No download required. The rs3 package bundles model weights and is installed automatically.

Usage

As a Python package

from crisprdesigner2 import (
    extract_guides_from_bed,
    apply_filters,
    get_passing_guides,
    score_guides_rs3,
    score_guides_guidescan,
    write_outputs,
)

# Extract guides from regions
guides = extract_guides_from_bed("regions.bed", "genome.fa")
import pandas as pd
guides_df = pd.DataFrame(guides)

# Filter by sequence quality
guides_df = apply_filters(guides_df)
passed = get_passing_guides(guides_df)

# Score on-target efficiency
scored = score_guides_rs3(passed, tracr="Chen2013")

# Score off-target specificity (optional)
scored = score_guides_guidescan(scored, "guidescan2.bam.sorted", "hg38")

# Write outputs
write_outputs(scored, "output_dir")

Public API

from crisprdesigner2 import (
    # Extraction
    extract_guides,              # Extract from single region
    extract_guides_from_bed,     # Extract from BED file
    reverse_complement,
    CONTEXT_PADDING,

    # Filtering
    apply_filters,               # Apply all sequence filters
    get_passing_guides,          # Get guides passing all filters
    is_valid_guide,              # Check single guide
    FILTERS,                     # Dict of filter functions

    # Scoring
    score_guides_rs3,            # RS3 on-target scoring (batch)
    score_single_guide_rs3,      # RS3 scoring (single guide)
    score_guides_guidescan,      # GuideScan2 off-target scoring

    # Output
    write_outputs,               # Write .txt and .bed files
    read_design_guides_txt,      # Read existing output

    # Caching
    load_predesigned_guides,     # Load cached guides
    merge_with_predesigned,      # Merge new + cached guides
)

As a Snakemake workflow

Configure config/config.yaml:

genome: "hg38"
regions: "path/to/regions.bed"
genome_fasta: "path/to/genome.fa"
guidescan2_index: "path/to/guidescan2.bam.sorted"
output_dir: "results/GuideDesign"
predesigned_guides: ""  # Optional: path to cached guides

Run the workflow:

# Dry run (validation)
snakemake -n --configfile config/config.yaml

# Full run
snakemake --configfile config/config.yaml --cores 4

Output files

designGuides.txt

Tab-separated file with scored guides:

Column	Description
chr	Chromosome
start	Start position (0-based)
end	End position (exclusive)
locus	Formatted locus string
strand	Strand (+ or -)
GuideSequenceWithPAM	23-nt sequence (protospacer + PAM)
guideSet	Region name from input BED
RS3_score	On-target efficiency (log-odds, typically -2 to +2)
specificity_score	Off-target specificity (0-1, higher = better)

designGuides.bed

BED6 format for genome browser visualization.

Interpreting scores

RS3 on-target scores

RS3 scores are log-odds values, typically ranging from -2 to +2. Higher scores indicate better predicted on-target efficiency.

GuideScan2 specificity scores

Specificity scores range from 0-1 (higher = more specific / fewer off-targets). The GuideScan2 paper recommends specificity score > 0.2 as a cutoff (Perez et al., Genome Biology 2025).

Guides with NaN specificity either:

• Are not in the reference genome
• Have multiple perfect matches in the genome

Sequence filters

Filter	Criterion	Reason
Pol III termination	>1 T in last 4 nt	Early transcription termination
High T/U content	>40% T	Reduced guide stability
T/U homopolymer	4+ consecutive T	Pol III termination
Mononucleotide repeat	5+ consecutive same base	Synthesis/sequencing issues
Low complexity	Various repeat patterns	Off-target concerns
Low GC	≤20% GC	Unstable RNA structure
High GC	≥90% GC	Synthesis issues

Project structure

CRISPRDesigner2/
├── pyproject.toml                      # Package metadata
├── src/
│   └── crisprdesigner2/                # Python package
│       ├── __init__.py                 # Public API
│       ├── extraction.py               # Guide extraction
│       ├── filters.py                  # Sequence filters
│       ├── scoring.py                  # Combined scoring exports
│       ├── _rs3_scoring.py             # RS3 implementation
│       ├── _guidescan_scoring.py       # GuideScan2 implementation
│       ├── output.py                   # Output file generation
│       └── cache.py                    # Pre-designed guide caching
├── Snakefile                           # Workflow definition
├── config/
│   └── config.yaml                     # Configuration template
├── workflow/
│   ├── envs/
│   │   └── CRISPRDesigner2.yaml        # Conda environment
│   └── scripts/
│       ├── snakemake_*.py              # Snakemake rule scripts
│       └── *.py                        # Thin wrappers (backward compat)
├── tests/
│   ├── data/                           # Test fixtures
│   └── test_*.py                       # Test modules
└── README.md

Testing

conda activate CRISPRDesigner2

# Run all tests
python -m pytest tests/ -v

# Run specific module
python -m pytest tests/test_guide_extraction.py -v

Troubleshooting

Import errors

Ensure the package is installed:

conda activate CRISPRDesigner2
pip install -e .

Missing GuideScan2

If GuideScan2 is unavailable, specificity scores will be NaN. The workflow still completes.

Slow GuideScan2 queries

Ensure the BAM index exists:

ls path/to/guidescan2.bam.sorted.bai
# If missing:
samtools index path/to/guidescan2.bam.sorted

License

See LICENSE file.

Citation

If you use this tool, please cite:

• RS3: Doench et al., Nature Biotechnology, 2016
• GuideScan2: Perez et al., Genome Biology, 2025