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Fluorescence measurement in CONAN and the Cas12a gate

ZN Zhou Nie

Last updated date: Mar 11, 2021 Views: 812 Forks: 0

original An abbreviated version of this protocol was published in Science Advances
A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics
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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

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

 

ReagentVolumeFinal Concentration
2 mM gRNA5 μL500 nM
5 mM LbCas12a2 μL500 nM
10 mM MgCl22 μL1 mM
DEPC water11 μL 
TOTAL VOLUME20 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:

 

ReagentVolumeFinal Concentration
500 nM LbCas12a/gRNA2 μL50 nM
5× CRISPR buffer4 μL1×

various concentrations of

Target dsDNA

1 μL 
20 mM FQ reporter1 μL1 mM
DEPC water12 μL 
TOTAL VOLUME20 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 QuantStudioTM 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

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

 

ReagentVolumeFinal Concentration
2 mM gRNA-T5 μL500 nM
5 mM LbCas12a2 μL500 nM
10 mM MgCl22 μL1 mM
DEPC water11 μL 
TOTAL VOLUME20 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:

 

ReagentVolumeFinal Concentration
500 nM LbCas12a/gRNA2 μL50 nM
5× CRISPR buffer4 μL1×

various concentrations of

Target dsDNA

1 μL 
10 mM scgRNA2 μL1 mM
10 mM aDNA1 μL500 nM
5 mM LbCas12a4 μL1 mM
DEPC water6 μL 
TOTAL VOLUME20 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 QuantStudioTM 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.

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Nie, Z(2021). Fluorescence measurement in CONAN and the Cas12a gate. Bio-protocol Preprint. bio-protocol.org/prep924.
  2. Shi, K., Xie, S., Tian, R., Wang, S., Lu, Q., Gao, D., Lei, C., Zhu, H. and Nie, Z.(2021). A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Science Advances 7(5). DOI: 10.1126/sciadv.abc7802

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