Cas12 Guide Finder

How SPACER identifies and extracts candidate guide RNAs for Cas12 (DNA-targeting) enzymes.

Overview

Cas12 guide finding is a PAM-dependent process. SPACER scans both strands of the input DNA sequence for valid PAM motifs, then extracts the adjacent spacer sequence from the non-target strand. Each extracted spacer becomes a candidate guide that proceeds to scoring.

PAM Scanning

The PAM (Protospacer Adjacent Motif) is a short sequence required for Cas12 enzyme recognition. SPACER scans the input sequence using IUPAC-aware pattern matching, supporting degenerate bases in PAM definitions. For Cas12a, the PAM is located on the 5′ side of the protospacer on the non-target strand.

PAM	Enzyme	Description
TTTN	AsCas12a	Three T's followed by any nucleotide (default)
TTTV	LbCas12a	Three T's followed by A, C, or G (excludes TTTT)

Both the forward and reverse complement strands are scanned. When a PAM is found on the reverse complement, the corresponding spacer is extracted and reverse-complemented so that all reported spacers read 5′→3′ on the non-target strand.

Spacer Extraction

Once a PAM is identified, SPACER extracts the spacer sequence immediately downstream (3′) of the PAM on the non-target strand. The extraction length is configurable:

Parameter	Range	Default
Spacer length	18–24 nt	20 nt

If the remaining sequence downstream of a PAM is shorter than the configured spacer length, that site is skipped. The extraction position is recorded so guides can be mapped back to their genomic coordinates.

Strand Handling

Cas12 targets double-stranded DNA, so both strands must be scanned. SPACER internally generates the reverse complement of the input and runs PAM scanning on both orientations. The result set includes guides from both strands, each annotated with its strand of origin (forward or reverse) and its 1-based start position on the input sequence.

Info

All Cas12 spacer sequences are reported in the non-target strand orientation (5′→3′) using DNA notation (T, not U). When a direct repeat is configured, the crRNA field contains the full guide in RNA form (U instead of T). The spacer sequence itself is always DNA — suitable for direct use in oligo ordering.

Deduplication

When scanning both strands, the same spacer sequence may appear at multiple positions. SPACER deduplicates candidates by their sequence context — identical spacers with the same flanking context are collapsed into a single candidate. The total number of pre-dedup hits is tracked internally but only unique candidates proceed to scoring.

Tip

Use TTTV for the most stringent selection (LbCas12a). If you need more candidates, switch to TTTN which includes TTTT sites — but note that TTTT PAMs may yield guides with lower in vivo activity.