Part:BBa_K3136004

FnCas12a-typeIIS

It is a conding sequence of CRISPR-Cas12a from Francisella novicida U112. The sequence is coden optimized.

Methods:

1. Put the EP tube containing purified FnCas12a protein at 99℃ water for 15 minutes.

2. After heating, centrifuge the tubes for 5min.

3. Take and install the gel, add the electrophoresis buffer to the sample hole, and check if there is any leakage. Add marker, to the first and last sample holes, add FnCas12a to the other sample holes, respectively.

7. Connect the electrophoresis device to the power supply. Connect the positive electrode to the tank and the negative electrode to the slot for electrophoresis. The voltage is adjusted to 160V.

8. Turn off the power supply and disconnect the electrode until the bromophenol blue reaches the bottom of the release adhesive. Remove the glass sheet from the electrophoresis device and then remove the gel.

9. Soak the gel in Coomassie brilliant blue dye and dye it in a horizontal shaking bed for 15 min.

Observe the protein bands

Cas12a detection method:

Materials:

Water. 14.5 μL

Buffer. 2 μL

Enzyme (Cas12a). 0.5 μL

Template. 0.5 μL

(From solution we made after LAMP)

Detector. 2 μL

crRNA. 0.5 μL

(Note: the Deteror is sequence of ssDNA HEX-N12-BHQ1 or FAM-N12-BHQ1 or FITC-T14-Biotin)

Procedure:

1. Add all the materials together:

• Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.

• Secondly, use pipette to straw 2 μL buffer to the previous tube.

• Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.

• Next, use pipette to straw 0.5 μL template to the previous tube.

• Then, add 0.5 μL crRNA to the previous tube.

• Finally, use pipette to straw 2 μL detector to the previous tube.

2. Straw 1 μL of the mixed solution in the tubes to the 96-well Plate.

3. Detect the solution using fluorescent detector machine to check the fluorescence when they react 10min.

Results:

We used two types of fluorescent probe (FAM and HEX) to check the fluorescence of FnCas12a reaction system at 10min as shown in Fig1. Linked to the control and negative samples, the positive sample has a high fluorescence. It means that FnCas12a slices the fluorescent probe in the reaction system and the cracked fluorescent probe produce fluorescence. we also checked the fluorescence of FnCas12a reaction system at 10min by lateral flow using briDetect (Milenia Biotec GmbH) (Fig2).

Fig1. The fluorescence of FnCas12a reaction system at 10min

Fig2. The fluorescence of FnCas12a reaction system by briDetect

Using fluorescent detector machine, we can detect the fluorescence of FnCas12a reaction system at different time by setting up program properly in the machine. We detect the fluorescence under different concentration FnCas12a and different time(Fig3). With the time growth, the fluorescence gradually raised until a certain time. Higher concentration FnCas12a had higher fluorescence and the fluorescence increased quickly.

Fig3. The fluorescence of FnCas12a reaction system in different time.

Reference

Li S Y, Cheng Q X, Liu J K, et al. CRISPR-Cas12a has both cis-and trans-cleavage activities on single-stranded DNA[J]. Cell research, 2018, 28(4): 491.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 310
  Illegal PstI site found at 364
  Illegal PstI site found at 1627
  Illegal PstI site found at 2533
  Illegal PstI site found at 3727
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 310
  Illegal PstI site found at 364
  Illegal PstI site found at 1627
  Illegal PstI site found at 2533
  Illegal PstI site found at 3727
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1253
  Illegal BglII site found at 1586
  Illegal BglII site found at 1706
  Illegal BglII site found at 2000
  Illegal BglII site found at 2444
  Illegal BglII site found at 3335
  Illegal BglII site found at 3425
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 310
  Illegal PstI site found at 364
  Illegal PstI site found at 1627
  Illegal PstI site found at 2533
  Illegal PstI site found at 3727
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 310
  Illegal PstI site found at 364
  Illegal PstI site found at 1627
  Illegal PstI site found at 2533
  Illegal PstI site found at 3727
  Illegal NgoMIV site found at 2480
  Illegal NgoMIV site found at 3319
  Illegal NgoMIV site found at 3530
  
• 1000
  COMPATIBLE WITH RFC[1000]