This tool designs sgRNAs for SpCas9-mediated genome editing based on the standard academic pipeline:
1. PAM Recognition
Targets are identified by the 5'-NGG-3' Protospacer Adjacent Motif (PAM). The 20bp sequence upstream of the PAM is extracted as the spacer.
2. Specificity Check
Specificity is verified using Local BLASTn against the selected Rosa genome.
High Specificity means the target sequence is unique in the entire genome (Hits=1).
3. GC Content
Ideally between 30% - 80%. Extreme GC content may affect transcription efficiency or binding stability.
4. Poly-T Filter
Sequences containing TTTT are flagged/excluded as they act as transcription terminators for Pol III promoters (e.g., U6).