Purpose: Reassemble Cas12a-sgRNA compound and test if the system can recognize the target sequences in the form of plasmids, oligo DNAs and bacterium after lysis.

8.1.1. Electrophoresis test

  1. Prepare oligoDNA groups:
    Materials needed:
    oligo DNA concentration /ng/μl oligo DNA volume/μl sgRNA volume/μl cas12a protein volume/μl reaction buffer volume/μl
    ipaH 4.9 20.4 5 3 21.6
    invA 64.95 1.54 5 3 40.46
    16S 0.925 0.925 5 3 41.075
    cagA 2.59 2.59 5 3 39.41

    *Note that the volume of oligo DNA varies according to its concentration. But each centrifuge tube should hold a total of 100M oligo DNA. The reaction buffer will also vary to achieve a fixed volume of 50 μl.

    1. In 8 separate 200μL centrifuge tube, add in sgRNA (cagA sgRNA1, cagA sgRNA2, ipaH sgRNA1, ipaH sgRNA2, invA sgRNA1, invA sgRNA2, 16S sgRNA1, 16S sgRNA2) cas12a protein and reaction buffer in the following volumes using a pipette in a clean bench.
    2. Centrifuge the mixture for 30s.
    3. Incubate the solution in a gradient thermal cycler at 37℃ for 10 minutes.
    4. In each centrifuge tube, add in the corresponding oligo DNA according to the volume recorded in the table above using a pipette.
    5. Centrifuge the systems briefly.
    6. In 4 separate centrifuge tubes, add in 50μl of ipaH, invA, cagA ,and 16S oligo DNA without incubation as the controlled group.
    7. Incubate all tubes in a gradient thermal cycler at 37℃ for 2 hours and then at 95℃ for 5 minutes to denature Cas12a proteins.
  2. Electrophoresis of all 8 oligo DNA tubes:
    1. Add 2μl 10×Loading Buffer to each 20μl Oligo DNA system
    2. Load 10μl 10× Buffer and Oligo DNA mixture into each well
    3. Load 10μl DNA marker in the well
    4. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. This will take about 20 minutes
    5. Open UV light to visualize DNA fragments

8.2.1. Using ssDNA (fluorescent) and multiscan ascent

  1. Prepare plasmids groups:
    Materials needed:
    plasmid vol/μL ssdna volume/μL sgRNA volume/μL cas12a protein volume/μL reaction buffer volume/μL
    ipaH 0.5 3 5 3 38.5
    invA 0.5 3 5 3 38.5
    16S 0.5 3 5 3 38.5
    cagA 0.5 3 5 3 38.5
    1. In 8 separate 200μL centrifuge tubes, add in sgRNA (cagA I, cagA II, ipaH I, ipaH II, invA I, invA II, 16S I , 16S II), cas12a protein and reaction buffer in the volumes depicted in the above chart using a pipette in a clean bench.
    2. Incubate the solution in a gradient thermal cycler at 37℃ for 10 minutes.
    3. In each centrifuge tube, add in the corresponding plasmids and ssDNA according to the volume recorded in the table above.
    4. Incubate the tubes in a gradient thermal cycler at 37℃ for 2 hours and then at 95℃ for 5 minutes to denature Cas12a proteins.
  2. Configure Negative Control Group of Cas12A-crRNA complex (without incubation):
    1. Add 38.5μl reaction buffer and 3μl of 50nM Cas12a to a microtube.
    2. Divide the reagent into 8 micro tubes of 5μl each (the excess will be used as a backup)
    3. 5μl of corresponding sgRNA (cagA sgRNA1, cagA sgRNA2, invA sgRNA1, invA sgRNA2, 16S sgRNA1, 16S sgRNA2, ipaH sgRNA1, ipaH sgRNA2) were added into each tube
    4. 0.5μl of corresponding plasmid and 3μl ssDNA fluorescent probe were added in every tube
  3. Measurement of ssDNA fluorescence value:
    1. Pipette 37 μl of solution from each of the tubes and controlled group* into the 96 wells light screen
    2. Measure fluorescence response of the bacteria culture using Multiskan Ascent

8.3.1. Test the efficacy of Cas12a-sgRNA system using electrophoresis:

  1. Bacteria lysis
    1. Using a pipette, transfer 1 ml of each of the four E. coli cultures(16S, cagA, ipaH, invA) into a centrifuge tube and centrifuge for 1 minute at 8000 rpm. Discard the supernatant.
    2. Add 50 μLllysis buffer into all four tubes.
    3. Place all tubes into a water bath and heat at 80℃ for 10 minutes
    4. Centrifuge all tubes again at 8000 rpm for 2 minutes
    5. Pipette at least 5 μl for each of the four E. coli colonies into at least two separate 20 μl microtubes. A total of 14 tubes of the bacterium are collected in this step.
    6. Extract 10 μl of each of the four E. coli inoculants into a microtube using a pipette and store them in a 4°C environment to use for electrophoresis in step 4.
  2. Incubation of Cas12a and sgRNA system and cleavage of target sequences
    1. A total of 16 1.5ml microtubes (1 tube for each sgRNA and the other as the controlled group) will be prepared each with 45μl of solution. Add all the following reagents and mediums using a pipette and on a clean bench.
      • Cas12a protein (3 μl)
      • 50/500ng sgRNA (5μl)
      • Reaction buffer (37 μl)
    2. Incubate in a gradient thermal cycler at 37℃ for 10 minutes.
    3. In all 14 microtubes, add in 5 μl of each of the four corresponding bacteria and label the tubes.
    4. Place one complete set of tubes with non-repetitive sgRNA sequences (8 tubes) in a gradient thermal cycler and PCR at 12000×g for 5 minutes.
    5. Centrifuge the tube at 8000×g, allowing the bacterium fully interact with the system.
    6. Incubate the product at 37℃ for 2 hours.
    7. Further heat the tubes at 95 °C for 5 minutes to halt all denature all Cas12a proteins and halt all protein activities.
  3. PCR
    1. Add 60 μl Mix Master Enzyme, 42 μl ddH2O, 12 μl Primer (6 μl Forward Primer and 6 μl Reverse Primer) and 6 μl incubated medium from step 2 into a centrifuge tube for each of the 14 tubes and run PCR in a gradient thermal cycler.
  4. Prepare electrophoresis
    1. Add in the following reagents using a pipette into a beaker and briefly heat it until all reagents fully dissolve
      • 2 g Agarose Regular
      • 100 ml TAE solution
      • 3 μl Nucleic Acid Gel Stain
    2. Add gel into the mould and wait until it solidifies.
    3. Add TAE buffer and gel into the electrophoresis machine
  5. Pipette 10 μl of 18 bacterial samples into each of the gel’s wells and run electrophoresis at 180V for 10 minutes.

8.3.2. Test the efficacy of the Cas12a-sgRNA system using ssDNA (fluorescent) and multiscan ascent

  1. Bacteria lysis
    1. Using a pipette, transfer 1 ml of each of the four E. coli cultures into two centrifuge tubes and centrifuge for 1 minute at 8000 rpm. Discard the supernatant.
    2. Add 50 μl lysis buffer into all four tubes.
    3. Place all tubes into a water bath and heat at 80℃ for 10 minutes
    4. Centrifuge all tubes again at 8000 rpm for 2 minutes
    5. Obtain 2 μl of the supernatant and pipette 4 μL for each of the two E. coli colonies into two separate 20 μl microtubes.
  2. Incubation of Cas12a and sgRNA system and target sequence cleavage
    1. A total of 16 1.5ml microtubes (2 tubes for each plasmid and 8 as controlled) will be prepared each with 50μl of solution. Add all the following reagents and mediums using a pipette and in a clean bench.
      • Cas12a protein (3 μl)
      • 50/500ng sgRNA (5μl)
      • Reaction buffer (34 μl)
    2. Incubate in a gradient thermal cycler at 37℃ for 10 minutes.
    3. To 8 of the 1.5 ml microtubes, add in 2 μl of the corresponding bacterium supernatant from step 1. Leave the other tubes bacteria-free.
    4. Incubate all tubes at 37℃ for 2 hours in a gradient thermal cycler.
    5. Further heat all tubes at 95℃ for 5 minutes to halt protein activities.
  3. Test using ssDNA and multiscan accent
    1. In a 96 well light screen, add 3 μl of fluorescent ssDNA and 2 μl of 3 ipaH E. coli plasmids (1 well for each of the two sgRNAs and 1 well without the plasmid as control), filling a total of 8 wells. In the four wells that are not the controlled group, add in Cas12a-sgRNA assay until the volume meets 50 μL.
    2. Test for fluorescent using the multiscan accent every 0.5 hours for the next 2 hours. Results are depicted in the table and graph below.