Enzyme Families

SPACER supports two CRISPR enzyme families optimized for nucleic acid detection.

Cas12 vs Cas13

Cas12 (DNA-Targeting)

Cas12 enzymes (primarily Cas12a/Cpf1) are RNA-guided DNA endonucleases. They recognize a T-rich PAM (Protospacer Adjacent Motif) upstream of the target site. Upon binding, they cleave the target dsDNA and then exhibit collateral cleavageactivity on nearby ssDNA, which is exploited for signal amplification in DETECTR³-style assays.

In SPACER, Cas12 guide design requires specifying a PAM sequence. The spacer is extracted from the non-target strand, reading 5′→3′ downstream of the PAM. AI activity prediction for Cas12 uses the EasyDesign model¹, which was trained specifically on LbCas12a activity data.

Cas13 (RNA-Targeting)

Cas13 enzymes are RNA-guided RNA-targeting nucleases. They do not strictly require a PAM, though some variants show preference for certain PFS (Protospacer Flanking Sequence) motifs. Upon binding the target RNA, Cas13 activates non-specific collateral cleavage of nearby ssRNA — target cleavage itself is not required to trigger this activity. This collateral effect is the basis for the SHERLOCK⁴ diagnostic platform.

Cas13 guide design in SPACER can optionally incorporate PFS preferences and benefits from the ADAPT AI activity prediction model², which was trained specifically on Cas13a (LwaCas13a) activity data.

Cas13 Variants

SPACER supports three characterized Cas13 orthologs, each with distinct PFS (Protospacer Flanking Sequence) preferences that affect scoring:

Variant selection affects the PFS scoring penalty applied during the heuristic quality component of the assay score. A Generic variant option is also available when no specific PFS filtering is desired.