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. 2022 Jun 28;3:e9. doi: 10.1017/qrd.2022.7

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© The Author(s) 2022

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.

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Figure 1. — Michaelis–Menten kinetics model of trans-cleavage of activated CRISPR-Cas12. The Cas12 (blue blob) is functionalised with a synthetic guide RNA molecule (shown here as a hairpin structure). In this rate-limiting trans-cleavage step, the enzyme has been functionalised by recognition and cleavage of the target DNA molecule (cleaved ssDNA target shown as a small blue strand). The enzyme indiscriminately cleaves ssDNA. By design, stoichiometry then favours cleaving of nucleic acid reporter probes functionalised with a fluorophore (F) and quencher (Q). is the Michaelis–Menten constant defined in terms of the off-rate k_r, turnover rate and forward rate , as shown in this figure.

Inline graphic — Michaelis–Menten kinetics model of trans-cleavage of activated CRISPR-Cas12. The Cas12 (blue blob) is functionalised with a synthetic guide RNA molecule (shown here as a hairpin structure). In this rate-limiting trans-cleavage step, the enzyme has been functionalised by recognition and cleavage of the target DNA molecule (cleaved ssDNA target shown as a small blue strand). The enzyme indiscriminately cleaves ssDNA. By design, stoichiometry then favours cleaving of nucleic acid reporter probes functionalised with a fluorophore (F) and quencher (Q). is the Michaelis–Menten constant defined in terms of the off-rate k_r, turnover rate and forward rate , as shown in this figure.