PAM Sequences
Understanding Protospacer Adjacent Motifs and their role in guide RNA extraction.
What is a PAM?
A PAM (Protospacer Adjacent Motif) is a short DNA sequence adjacent to the target site that is required for Cas enzyme recognition. The PAM is present in the target DNA but not in the guide RNA. Without a matching PAM, the Cas enzyme cannot bind and cleave the target.
For Cas12a enzymes, the PAM is located on the 5′ side of the protospacer (upstream). SPACER scans the input sequence for PAM occurrences and extracts the adjacent spacer region as a guide candidate.
Supported PAM Sequences
| PAM | IUPAC Expansion | Matching Bases | Stringency |
|---|---|---|---|
| TTTN | TTT[ACGT] | TTTA, TTTC, TTTG, TTTT | Standard (default) |
| TTTV | TTT[ACG] | TTTA, TTTC, TTTG | Stringent (excludes TTTT) |
TTTV > TTTN. The fourth base matters — TTTA and TTTG tend to produce higher activity than TTTT.PAM vs PFS
PAM and PFS are fundamentally different mechanisms. A PAM (Protospacer Adjacent Motif) is required for Cas12/Cas9 protein binding to DNA — without a matching PAM, the enzyme cannot engage the target. In contrast, a PFS (Protospacer Flanking Sequence) is a positional preference that influences Cas13 cleavage efficiency on RNA. PFS is not strictly required for binding, but unfavorable flanking nucleotides reduce activity.
PFS for Cas13
Different Cas13 orthologs have different PFS preferences at the target RNA flanking positions. SPACER evaluates PFS based on the selected Cas13 variant and applies a scoring penalty within the heuristic quality component when an unfavorable PFS is detected.
| Variant | PFS Position | Avoid | Preferred (IUPAC) |
|---|---|---|---|
| LwaCas13a | 3′ of target | G | H (A, C, or U) |
| LbuCas13a | 3′ of target | G | H (A, C, or U) |
| PsmCas13b | 5′ of target | C | D (A, G, or U) |
| Generic | — | — | No PFS filtering applied |