transcleavage

Transcleavage, or trans-cleavage, is a term used to describe a cleavage event that occurs on substrates other than the enzyme’s primary target, i.e., cleavage in trans rather than cis. In CRISPR-associated nuclease systems, transcleavage refers to collateral nuclease activity: after the Cas effector complex binds its target sequence, the nuclease becomes activated and cleaves non-target nucleic acids present in the reaction mixture. This property is exploited in diagnostic assays because cleavage of reporter molecules produces a detectable signal.

Cas12 family nucleases (such as Cas12a) show trans-cleavage of single-stranded DNA (ssDNA) once activated by target binding; Cas13 family nucleases (such as Cas13a) cleave non-target RNA reporters in trans after recognizing a target RNA. These activities underpin many CRISPR-based diagnostics, including platforms like DETECTR (Cas12-based) and SHERLOCK (Cas13-based). The magnitude and kinetics of transcleavage depend on enzyme concentration, guide RNA, target sequence, and reaction conditions, and are generally considered a tradeoff between sensitivity and potential off-target effects.

Transcleavage is distinct from cis-cleavage, where the target molecule itself is cleaved at or near the intended site. In the literature, the term transcleavage is sometimes used interchangeably with collateral cleavage, though some authors use collateral cleavage to emphasize cleavage of non-target substrates in trans.

Limitations and applications: while advantageous for rapid detection, transcleavage-based assays require careful design to minimize unintended cleavage and to ensure robust signal discrimination in complex samples. Ongoing research seeks to broaden the range of nucleic acid substrates and to improve specificity and throughput.