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bioRxivMolecular BiologyPreprint

Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

Ryan T. FuchsiD, Jennifer L. CurcuruiD, Megumu Yamada Mabuchi, Paul YourikiD, G. Brett RobbiD

PDFDOI

Abstract summary

Claude Opus 5.5 · xhigh effort · 18 Oct 2026 · from the abstract · CC BY-NC-ND 4.0

Once activated, Cas12a degrades RNA and double-stranded DNA in trans, not only single-stranded DNA, with the amount depending on buffer, guide RNA features and which ortholog is used.

Cas12a orthologs are repurposed for genome editing in non-native organisms and for in vitro DNA manipulation, and activation by guide RNA-target DNA pairing was already known to trigger non-specific single-stranded DNA degradation in trans. Reporting that the same activated state also attacks RNA and double-stranded DNA, with a magnitude that varies with buffer, guide RNA and ortholog, changes what counts as a safe assumption in those uses.

Key results

  1. SuggestedTrans (collateral) nuclease activity of activated Cas12a is not confined to single-stranded DNA: RNA and double-stranded DNA substrates are also cut, which becomes clearer once the reaction buffer is adjusted.

    Reported from in vitro cleavage experiments; the abstract gives no cleavage rates, substrate concentrations or relative efficiencies across substrate types. · Abstract

  2. SuggestedHow much trans cleavage occurs depends on the guide RNA, specifically the base composition of the target sequence and the guide length.

    Stated as a trend across guides; no per-guide measurements or sequence rules are given in the abstract. · Abstract

  3. ClaimedTrans activity differs among the three most studied Cas12a orthologs, with the enzyme from Lachnospiraceae bacterium ND2006 described as the most active.

    A ranking is asserted with the hedge that this ortholog appears most active; no activity values or ortholog-by-ortholog comparison numbers appear in the abstract. · Abstract

Methods, data and reproducibility

In vitro cleavage assays with Cas12a activated by guide RNA-target DNA pairing, testing single-stranded DNA, double-stranded DNA and RNA as trans substrates while varying reaction buffer composition, guide RNA target sequence and length, and the Cas12a ortholog.

  • In vitro trans-cleavage assays across substrate types, buffers and guide RNAs
  • Comparison of the three most studied Cas12a orthologs, including the enzyme from Lachnospiraceae bacterium ND2006 · Lachnospiraceae bacterium ND2006 (one of three orthologs) · n = 3 orthologs
Methods & tools
Cas12a (Cpf1), guide RNA
Limitations (2)
  • Everything is reported qualitatively: no rates, specific activities or substrate preferences are quantified in the abstract, so the size of the RNA and double-stranded DNA effect is unknown. · noted in this summary
  • The work is in vitro and buffer-dependent, so whether the broadened trans activity matters inside cells or in applied in vitro workflows is not addressed. · noted in this summary

Read this if you use Cas12a for in vitro DNA manipulation or genome editing and need to know which substrates an activated enzyme will also degrade, and how to tune that.

Citations
51

Abstract

CRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulation in vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation in trans, after single turnover on-target cleavage in cis. We find that the non-specific trans nuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of the trans nuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude of trans nuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a from Lachnospiraceae bacterium ND2006 appears to be the most active.

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