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.PFS for Cas13
Cas13 enzymes do not require a PAM in the traditional sense. However, some Cas13 variants show a preference for certain PFS (Protospacer Flanking Sequence) motifs at the 3′ end of the target.
SPACER handles PFS scoring automatically — there is no user-configurable PFS setting. The engine evaluates the flanking nucleotide at the 3′ end of each candidate guide and applies a scoring penalty when an unfavorable PFS is detected (e.g., a 3′ G for LwaCas13a). This adjustment appears as the PFS adjustment component in the score breakdown.