sg5 sgRNA Designer

A command-line tool for designing CRISPR sgRNAs targeting Drosophila melanogaster genes using the SpCas9/NGG system.

Fetches gene sequences directly from the Ensembl REST API, scans both strands for NGG PAM sites, scores each candidate guide, and outputs a ranked list.

Features

• Resolves gene symbols to Ensembl IDs automatically
• Fetches sequence with 200 bp flanking regions (for promoter-targeting guides)
• Scans both strands for NGG PAM sites
• Scores guides based on GC content, homopolymer runs, poly-T stretches, and 5' G preference
• Outputs ranked results to terminal and optionally to CSV

Requirements

• Python 3.10+
• requests

Install dependencies:

pip install requests

Usage

python sgrna_designer.py <gene_symbol> [--top N] [--output results.csv]

Examples

# Design top 20 guides for the white gene
python sgrna_designer.py white

# Get top 10 guides and save to CSV
python sgrna_designer.py rosy --top 10 --output rosy_guides.csv

Arguments

Argument	Description	Default
gene	Gene symbol (e.g. white, rosy, nos)	required
--top	Number of top guides to report	20
--output, -o	Output CSV filename	none

Scoring

Guides start at a score of 100 and are penalised for:

• GC% outside 40–70% (-20)
• Homopolymer runs ≥ 4 nt (-15)
• Poly-T stretch TTTT (-25, terminates U6 transcription)
• No 5' G (-5, U6 promoter preference)

Output

Results are printed to the terminal and optionally saved as a CSV with columns: guide_sequence, pam, strand, position_in_fetch, gc_percent, score, warnings.

Notes

• Designed for Drosophila melanogaster (dm6) and SpCas9 (NGG PAM)
• Requires internet access to query the Ensembl REST API
• Off-target analysis is not included — use tools like CRISPOR or Cas-OFFinder for that