Cas13 Guide Finder
How SPACER identifies and extracts candidate guide RNAs for Cas13 (RNA-targeting) enzymes.
Overview
Cas13 guide finding uses a sliding window approach rather than PAM scanning. Because Cas13 enzymes target single-stranded RNA and do not strictly require a PAM, SPACER generates candidate spacers by stepping through the target sequence one nucleotide at a time. All candidates proceed to scoring — PFS (Protospacer Flanking Sequence) preferences are applied as a scoring adjustment, not as a filter that removes candidates.
Sliding Window Extraction
SPACER moves a window of the configured spacer length across the target sequence, extracting every possible subsequence as a candidate guide. This yields the maximum number of candidates for downstream scoring and ranking.
| Parameter | Range | Default |
|---|---|---|
| Spacer length | 20–28 nt | 28 nt |
| Step size | Fixed | 1 nt |
For a target sequence of length L and spacer length S, the finder produces L − S + 1 candidate guides. Each candidate is recorded with its 1-based start position on the input.
DNA Input Handling
Cas13 targets RNA, but researchers often work with DNA sequences (e.g., from GenBank). SPACER accepts both DNA and RNA input. The reported spacer sequences are always in DNA form (using T, not U) for easy lookup against reference sequences. When a direct repeat is configured, SPACER assembles the full crRNA by prepending the direct repeat to the spacer and transcribing both to RNA (T → U). The crRNA field is 5′-[direct repeat]-[spacer]-3′, entirely in RNA form.
Cas13 Variant & PFS Evaluation
When configuring a Cas13 analysis, you can select a specific Cas13 variant (LwaCas13a, LbuCas13a, or PsmCas13b). The variant determines which PFS preferences are applied during scoring. PFS evaluation checks the flanking nucleotide at the appropriate position (3' or 5' depending on variant) and applies a penalty in the heuristic quality score for unfavorable flanking bases.
Single-Strand Processing
Unlike Cas12 (which scans both strands of dsDNA), Cas13 guide finding operates on a single strand. The input is treated as the target RNA, and spacers are extracted in the 5′→3′ direction. There is no reverse complement scanning, since Cas13 binds ssRNA directly.