Exonuclease-coupled real-time endonuclease activity assay

A composition when used for detecting activity of an endonuclease, the composition comprising a nucleic acid substrate for detecting activity of an endonuclease comprising:

• (a) a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair;
• (b) at least one structure on at least one nucleic acid strand inhibiting cleavage of the substrate by an exonuclease;
• and
• (c) a recognition sequence and a cleavage site for an endonuclease, wherein the endonuclease is selected from the group consisting of a nucleic acid-guided endonuclease, a zinc finger nuclease (ZFN), a ZFN conjugated to Fok I, a transcription activator-like effector nuclease (TALEN), an Argonaute endonuclease, an Arcus endonuclease, an engineered endoribonuclease;
• Cas3 and Cas7-11 and a Type II restriction endonuclease;
• wherein:
• the donor fluorophore and the acceptor fluorophore are not separated by the endonuclease cleavage site;
• and
• the composition further comprises the exonuclease.

A method for optimizing CRISPR endonuclease digestion reactions, the method comprising:

• (a) preparing a series of reaction mixtures with a CRISPR endonuclease, an exonuclease and a nucleic acid substrate comprising:
• i. a donor fluorophore and an acceptor fluorophore, said fluorophores forming a Fluorescence Resonance Energy Transfer (FRET) pair;
• ii. at least one structure on at least one strand inhibiting cleavage of the substrate by the exonuclease;
• and
• iii. a recognition sequence for an endonuclease;
• (b) contacting each of the reaction mixtures in the series with a series of nucleic acid targeting nucleic acids (NATNAs);
• (c) measuring fluorescence emitted by the reaction mixture, wherein a change in fluorescence indicates activity of the endonuclease;
• (d) selecting the NATNA yielding the highest fluorescence of the reaction mixture as the optimal NATNA.