PFS Scoring
How SPACER evaluates Protospacer Flanking Sequence preferences for Cas13 guide RNA design.
What Is PFS?
The Protospacer Flanking Sequence (PFS) is the nucleotide immediately adjacent to the protospacer on the target RNA. While Cas13 enzymes do not require a strict PAM like Cas12, experimental studies have shown that certain Cas13 variants exhibit higher cleavage activity when specific nucleotides are present at the 3′ flank of the protospacer.
PFS is sometimes described as the RNA equivalent of PAM, but the analogy is imperfect. Unlike PAMs, which are absolutely required for Cas12 target recognition, PFS preferences are statistical tendencies — a guide with an unfavorable PFS may still cleave its target, just with reduced efficiency.
PFS Preferences by Enzyme
| Enzyme Variant | PFS Position | Preferred | Avoided | Effect Size |
|---|---|---|---|---|
| LwaCas13a | 3′ | A, C, U (non-G) | G | ~2–4x activity difference |
| PsmCas13b | 3′ and 5′ | Non-C (3′), A/C (5′) | C (3′), G/U (5′) | ~2–3x |
| RfxCas13d (CasRx) | 3′ | Weak or none | — | Minimal |
The most well-characterized PFS effect is the 3′ non-G preference for LwaCas13a. Guides with a G at the 3′ PFS position show markedly reduced cleavage activity, likely because the G forms an unfavorable interaction with the Cas13 binding pocket during target recognition.
Scoring Function
SPACER automatically checks the nucleotide at the 3′ PFS position for Cas13 guides and assigns a score:
| 3′ PFS Nucleotide | Score | Interpretation |
|---|---|---|
| A | 1.0 | Optimal — strong activity expected |
| C | 1.0 | Optimal — strong activity expected |
| U | 1.0 | Optimal — strong activity expected |
| G | 0.0 | Unfavorable — significant activity reduction |
The binary scoring reflects the biological reality: the effect of a 3′ G is a substantial activity drop rather than a gradual decline. This is modeled as a hard penalty to strongly discourage selection of G-flanked guides when alternatives exist.
How SPACER Handles PFS
PFS scoring is applied automatically for all Cas13 analyses — there is no user-configurable setting. SPACER evaluates the 3′ flanking nucleotide of each candidate guide and applies a penalty when an unfavorable PFS is detected. The result appears as the PFS adjustment in the score breakdown.