We here upload the detailed protocol about the "Fluorescence measurement in CONAN and the Cas12a gate", and please see the attached file.

Fluorescence measurement in CONAN and the Cas12a gate

A. Run Cas12a gate detection

On ice, prepare Cas12a, gRNA, MgCl₂. Cas12a/gRNA complex can be prepared as follows:

Reagent	Volume	Final Concentration
2 mM gRNA	5 μL	500 nM
5 mM LbCas12a	2 μL	500 nM
10 mM MgCl₂	2 μL	1 mM
DEPC water	11 μL
TOTAL VOLUME	20 μL

2. Incubate LbCas12a and gRNA to generate LbCas12a/gRNA complex at 4 °C for 30 min. The purified and quantified LbCas12a is stored in the buffer [50 mM Tris (pH 8.0), 200 mM NaCl, 1 mM DTT, 50% glycerol] at -80°C.

3. The preparation of 1 mM dsDNA is similar to that of cgRNA.

4. The CRISPR buffer is [20 mM tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol, and heparin (50 mg/mL)].

5. On ice, prepare the LbCas12a gate reaction reagents.
The LbCas12a gate can be prepared as follows:

Reagent	Volume	Final Concentration
500 nM LbCas12a/gRNA	2 μL	50 nM
5× CRISPR buffer	4 μL	1×
various concentrations of Target dsDNA	1 μL
20 mM FQ reporter	1 μL	1 mM
DEPC water	12 μL
TOTAL VOLUME	20 μL

6. Place reactions on ice until ready to proceed.

7. Add the LbCas12a gate solution (20 mL) to a 96-well reaction plate (100 mL).

8. The fluorescence intensity tests were carried out on the QuantStudio^TM 7 Flex machine (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 4 hours with fluorescence measurements taken every 30 s. 6-Carboxy-fluorescein (FAM) was excited at 495 nm, and its emission at 520 nm was recorded.

B. Run CONAN detection

On ice, prepare LbCas12a, gRNA-T, MgCl₂.
LbCas12a/gRNA-T complex can be prepared as follows:

Reagent	Volume	Final Concentration
2 mM gRNA-T	5 μL	500 nM
5 mM LbCas12a	2 μL	500 nM
10 mM MgCl₂	2 μL	1 mM
DEPC water	11 μL
TOTAL VOLUME	20 μL

2. Incubate LbCas12a and gRNA to generate LbCas12a/gRNA complex at 4 °C for 30 min.

3. The preparation of 1 mM aDNA is similar to that of cgRNA.

4. On ice, prepare CONAN reaction reagents.
The CONAN can be prepared as follows:

Reagent	Volume	Final Concentration
500 nM LbCas12a/gRNA	2 μL	50 nM
5× CRISPR buffer	4 μL	1×
various concentrations of Target dsDNA	1 μL
10 mM scgRNA	2 μL	1 mM
10 mM aDNA	1 μL	500 nM
5 mM LbCas12a	4 μL	1 mM
DEPC water	6 μL
TOTAL VOLUME	20 μL

5. Place reactions on ice until ready to proceed.

6. Add the CONAN solution (20 mL) to a 96-well reaction plate (100 mL).

7. The fluorescence intensity tests were carried out on the QuantStudio^TM 7 Flex machine (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C for 4 hours with fluorescence measurements taken every 30 s. 6-Carboxy-fluorescein (FAM) was excited at 495 nm, and its emission at 520 nm was recorded.