tools-only Off-Target Prediction
Cas-OFFinder (CLI)
Cas-OFFinder searches genomes for potential off-target sites allowing mismatches.
# Input file format (input.txt):
# Line 1: Path to genome directory (2bit or fasta index)
# Line 2: PAM pattern (N = any, R = A/G, Y = C/T)
# Line 3+: Guide sequences with mismatch tolerance
# Example input.txt:
# /path/to/genome
# NNNNNNNNNNNNNNNNNNNNNGG
# ATCGATCGATCGATCGATCGNNN 4
# Run Cas-OFFinder
cas-offinder input.txt C output.txt # C = use CPU
cas-offinder input.txt G output.txt # G = use GPU (faster)
Cas-OFFinder Input Preparation
def prepare_cas_offinder_input(guides, genome_path, max_mismatches=4, pam='NGG'):
'''Prepare Cas-OFFinder input file
Args:
guides: List of 20nt guide sequences
genome_path: Path to genome directory with .2bit or indexed fasta
max_mismatches: Maximum mismatches to search (0-6 typical)
More mismatches = slower but more comprehensive
4 mismatches: good balance of speed and sensitivity
pam: PAM sequence (NGG for SpCas9)
'''
lines = [genome_path]
# Build pattern: 20 N's for guide + PAM
pattern = 'N' * 20 + pam
lines.append(pattern)
# Add each guide with mismatch tolerance
for guide in guides:
# Append NNN to represent PAM positions (not matched)
lines.append(f'{guide}NNN {max_mismatches}')
return '\n'.join(lines)
Parse Cas-OFFinder Output
import pandas as pd
def parse_cas_offinder_output(output_file):
'''Parse Cas-OFFinder results
Output columns:
- Guide: Query guide sequence
- Chromosome: Target chromosome
- Position: Genomic position (0-based)
- Sequence: Off-target sequence found
- Strand: + or -
- Mismatches: Number of mismatches
'''
columns = ['guide', 'chrom', 'position', 'sequence', 'strand', 'mismatches']
df = pd.read_csv(output_file, sep='\t', header=None, names=columns)
# Sort by mismatches (fewer = more concerning)
df = df.sort_values('mismatches')
return df
def summarize_off_targets(df):
'''Summarize off-target counts by mismatch number'''
summary = df.groupby(['guide', 'mismatches']).size().unstack(fill_value=0)
# Calculate specificity score
# Fewer off-targets with 0-2 mismatches = higher specificity
summary['specificity'] = 1 / (1 + summary.get(0, 0) * 100 +
summary.get(1, 0) * 10 +
summary.get(2, 0))
return summary
CFD Score Calculation
# Cutting Frequency Determination (CFD) score
# Predicts cleavage probability at off-target sites
# From Doench et al. 2016
# Position-specific mismatch penalties
CFD_MISMATCH_SCORES = {
# (position, ref_nt, target_nt): penalty_multiplier
# Position 1 = PAM-proximal, Position 20 = PAM-distal
# Values < 1 indicate reduced cutting
(1, 'C', 'A'): 0.5, (1, 'C', 'G'): 0.7, (1, 'C', 'T'): 0.3,
(1, 'G', 'A'): 0.4, (1, 'G', 'C'): 0.6, (1, 'G', 'T'): 0.3,
# ... (full matrix has all 20 positions x 12 mismatch types)
(20, 'C', 'A'): 0.9, (20, 'C', 'G'): 0.95, (20, 'C', 'T'): 0.85,
}
# PAM mismatch penalties
CFD_PAM_SCORES = {
'AGG': 0.26, 'CGG': 0.11, 'TGG': 0.02, # First position
'GAG': 0.07, 'GCG': 0.03, 'GTG': 0.02, # Second position
'GGA': 0.01, 'GGC': 0.01, 'GGT': 0.01, # Third position
}
def calculate_cfd_score(guide, off_target, pam='NGG'):
'''Calculate CFD score for an off-target site
CFD score interpretation:
- 1.0: Perfect match (on-target)
- >0.5: High probability of cleavage (concerning)
- 0.1-0.5: Moderate probability
- <0.1: Low probability (likely acceptable)
'''
score = 1.0
# Apply mismatch penalties
for i, (g, t) in enumerate(zip(guide, off_target[:20]), 1):
if g != t:
key = (i, g, t)
penalty = CFD_MISMATCH_SCORES.get(key, 0.5) # Default 0.5
score *= penalty
# Apply PAM penalty if not NGG
off_pam = off_target[20:23]
if off_pam != 'NGG' and off_pam in CFD_PAM_SCORES:
score *= CFD_PAM_SCORES[off_pam]
return score
Aggregate Off-Target Score
def calculate_guide_specificity(guide, off_targets):
'''Calculate aggregate specificity score for a guide
Uses sum of CFD scores for all off-targets.
Lower aggregate = more specific guide.
Specificity score interpretation:
- >0.9: Highly specific (excellent)
- 0.7-0.9: Specific (good)
- 0.5-0.7: Moderate specificity (acceptable)
- <0.5: Poor specificity (consider alternatives)
'''
cfd_sum = sum(calculate_cfd_score(guide, ot['sequence'], ot.get('pam', 'NGG'))
for ot in off_targets)
# Specificity = 1 / (1 + sum of off-target CFD scores)
specificity = 1 / (1 + cfd_sum)
return specificity
CRISPOR-style Analysis
def analyze_guide_specificity(guide_seq, genome_fasta, max_mm=4):
'''Full off-target analysis workflow
1. Run Cas-OFFinder to find potential sites
2. Calculate CFD scores for each site
3. Compute aggregate specificity
4. Flag high-risk off-targets
'''
import subprocess
import tempfile
# Prepare input
with tempfile.NamedTemporaryFile(mode='w', suffix='.txt', delete=False) as f:
f.write(f'{genome_fasta}\n')
f.write('N' * 20 + 'NGG\n')
f.write(f'{guide_seq}NNN {max_mm}\n')
input_file = f.name
output_file = input_file.replace('.txt', '_out.txt')
# Run Cas-OFFinder
subprocess.run(['cas-offinder', input_file, 'C', output_file], check=True)
# Parse results
off_targets = parse_cas_offinder_output(output_file)
# Calculate CFD for each
off_targets['cfd_score'] = off_targets.apply(
lambda row: calculate_cfd_score(guide_seq, row['sequence']), axis=1
)
# Flag high-risk (CFD > 0.5 and in exon)
# Would need exon annotations for full implementation
return off_targets
Related Skills
- genome-engineering/grna-design - Design guides before off-target check
- variant-calling/variant-annotation - Check if off-targets overlap known variants
- genome-intervals/bed-file-basics - Intersect off-targets with genomic features