CRISPR sgRNA Designer (Bio-chat Series)
This skill provides a rigorous workflow for designing and scoring CRISPR/Cas9 sgRNAs, specifically optimized for high-GC viral genomes where standard tools often struggle.
Key Capabilities
- Precision PAM Scanning: Automated scanning of target regions for SpCas9 (NGG) and alternative PAMs.
- GC-Weighted Scoring: Specialized weighting for high-GC templates (65-80% GC) to ensure thermodynamic stability of the R-loop.
- Positional Analysis: Calculates cut sites relative to functional features (ATG start codons, stop codons, or exon junctions).
- Off-target Defense: Rapid genomic indexing to ensure seed-region (12bp) uniqueness in small viral/microbial genomes.
- Donor Template Strategy: Automatically suggests synonymous mutations to prevent re-cutting of integrated donor sequences.
Workflow
- Target Identification: Provide a genomic region or gene name (e.g., PRV UL15 N-terminus).
- Sequence Extraction: Automatically pulls the latest reference sequences via NCBI E-utils.
- sgRNA Generation: Runs a sliding window scan for NGG PAMs within the target window.
- On-target Scoring:
- Position Weighting: Rewards specific nucleotides in the seed region.
- GC Penalty/Reward: Optimizes for the high-GC context of viruses like PRV.
- Result Ranking: Provides a ranked list of sgRNAs with comprehensive scoring (0-100) and experimental recommendations.
Technical Standards
- Scoring Model: Modified Rule Set 2 logic adapted for extreme sequence contexts.
- Seed Region: Strict 12bp specificity check.
- PAM Requirement: 5'-NGG-3' (Standard SpCas9).
Designed by ZJU PhD @ Bio-chat Community