CRISPR gRNA Scoring: On-Target vs Off-Target Heuristics Explained
GC content, seed-region composition, poly-T termination and PAM spacing: how CRISPR guide RNAs are scored, ranked and screened for off-target risk.
When selecting CRISPR guide RNAs, biophysical properties across the 20-nucleotide protospacer govern both cleavage efficiency and off-target cleavage.
**1. GC Content (40-65% Optimal):** Guides with <30% GC have unstable DNA:RNA heteroduplex binding, while guides with >75% GC fail due to secondary structure formation and difficult target strand displacement.
**2. The Seed Region (Positions 1-10 PAM-Proximal):** The 10 bases adjacent to the PAM sequence (NGG for SpCas9, TTTV for Cas12a) nucleate Cas9 binding. Single mismatches in this region drastically abort cleavage.
**3. Poly-T Termination:** Four or more consecutive Thymines (TTTT) trigger early transcription termination by RNA Polymerase III under U6 or H1 promoters. SciKeep automatically flags and penalizes poly-T motifs.
**4. Experimental Workflow:** Always select 3-4 top-ranked guides per target and empirically validate editing efficiency using ICE Sanger deconvolution or T7E1 endonuclease assays.