FQ reporter assays Notebook

Sunday,01/07/2018

This set of experiments are based on our protocol: Fluorophore-Quencher reporter Cas12a assay.

ts aim is to detect a specific dsDNA sequence (the Activator) with the CRSIPR/Cas12a system. If said sequence is found in the sample, Cas12a will unleash single-stranded DNase acivity and thus cleave every ssDNA found in the sample. We're using this mechanism to get a fluorescent read out using DNaseAlert (ssDNA reporters).

Our template is made of a non target strand (NTS) and a target strand (TS) (target 1_NTS /target 1_TS cf. supplementary materials of Chen, J. S. et al.)

NTS (non targeting strand): G C T T G T G G C C G T T T A C G T C G C C G T C C A G C T C G A C C A G G A T G G G C A C C A C C C C G G C

TS (targeting strand): G C C G G G G T G G T G C C C A T C C T G G T C G A G C T G G A C G G C G A C G T A A A C G G C C A C A A G C

The TS is the strand which will be recognized by the cas12a and thus is complementary to the crRNA.

We annealed our NTS and TS strand according to our annealing protocol: this is are activator/target.

We transcribed our crRNA using our transcription protocol from the following sequence:

G G T C G A G C T G G A C G G C G A C G A T C T A C A C T T A G T A G A A A T T A C C T A T A G T G A G T C G T A T T A A G

Monday 30/07/18

Testing Cas12a complex + DNaseAlert

Simple experience to test whether the CRISPR/cas12a system works (i.e. if the crRNA was correctly designed and did form a complex with the cas12a which should eventually cleave the ssDNA reporter (DNaseAlert).

To get optimal results we've mixed the protocols found in the Supplementary materials of the paper stated above and the protocol given with the lba cas12a from NEB

Protocol:

Final sample (20μl) : Cas12a (50nM), crRNA (62nM), activator (1nM), DNaseAlert (50nM), NEBuffer 10x (1x)

1. Add 2μl of nuclease free water
2. Add 2μl of NEBuffer
3. Add 1μl of Cas12a (1μM)
4. Add 1μl of crRNA (1.37 μM)
5. Incubate 10 minutes at room temperatures
6. Add 4μl of activator (5nM 4.a)
7. Incubate 10 minutes at 37°C
8. Add 10μl of DNaseAlert (single use tube diluted in 40μl)
9. Let incubate in 37°C
10. Look at the fluorescence at desired times (here we checked the fluorescence after ~10minutes)

Negative control (done at the same time as the sample)

1. Add 3μl of Nuclease free water
2. Add 2μl of NEBuffer
3. Add 1μl of crRNA
4. Incubate for 10 minutes at room temperature (with other sample)
5. Add 4 μl of activator
6. Incubate for 10 minutes at 37°C (with other sample)
7. Add 10μl of DNaseAlert (single use tube diluted in 40μl)
8. Look at the fluorescence at desired times (here we checked the fluorescence after ~10minutes)

Results

Analysis/Discussion

As it can be seen on the picture there are some fluorescence from the sample with the activator and none from the negative control (without cas12a). This suggest that our CRISPR/cas12a system activates correctly the DNaseAlert. BUT the negative control should have been the same as the other sample without the activator to see if cas12a truly only gains its nuclease activity when the activator is present in the sample.

Tuseday 31/07/18

Trial 1 (50 nM Cas12a, 62.5 nM crRNA, 50 nM DNaseAlert, NEBuffer 2.1)

This experiment contained 50 nM of DNaseAlert and negative control contained only DNaseAlert (in addition to buffer) Incubation step (30min/37°C) at the following concentrations: Lbcas12a 250nM / crRNA 320nM. We made different samples with different activator concentrations : 1nM-1μM.

Results:

Analysis:

Controls seem to work and the enzyme seems to be activated, since we can measure fluorescence in all samples. Also, those with higher concentrations of template DNA exhibit more fluorescence, except for the 100 nM sample which has a fluorescence higher than the 1 μM sample.

Discussion

Overall, controls seem to work, samples with higher concentrations of template DNA exhibit more fluorescence, except for the 100 nM sample which has a fluorescence higher than the 1μM sample. Regarding this, we could have inverted the samples. Another explanation would be that for higher concentration of activator, the enzyme might be cleaving that instead of DNaseAlert reporter. The negative control should be cas12a with crRNA without any template. We'll modify this for the next experiment.

Thursday 02/08/2018

Trial 2 (50 nM Cas12a, 62.5 nM crRNA, 100 nM DNaseAlert, NEBuffer)

We did this experiment using Nebuffer 2.1 protocol and added a negative control sample containing this time Cas12a enzyme. Incubation step (30min/37°C) at the following concentrations: Lbcas12a 250nM / crRNA 320nM. We made different samples with different activator concentrations : 1nM-1μM.

Results:

Analysis:

We can see the increase in the level of fluorescence signal (again 1 μM is below 100 nM activator in terms of fluorescence). Moreover, the negative control level is mostly above the ones with activators.

Discussion

It seems that Cas12a could be activated even without binding to the activator.. Or maybe we put some DNA by mistake. The pattern of fluorescence as a function of concentration is still not what we would expect (more fluorescence for concentrated samples). We’re planning on using Binding buffer for next trial.

Friday 03/08/2018

Trial 3 (50 nM Cas12a, 62.5 nM crRNA, 200 nM DNaseAlert)

We did this experiment as usual with the following changes: 200 nM DNaseAlert (concentration has been doubled), and again negative control containing the Cas12a enzyme. The experiment was performed using binding buffer (10X, 20mM Tris-HCl, pH7.5, 100mM KCL, 5mM MgCl2, 1mMDTT, 5% gylcerol, 50 ug/ml heparin) used in the paper.

Results:

Analysis

We can see the increase in the level of fluorescence signal (again 1 μM is below 100 nM activator in terms of fluorescence). This time the negative control has the lowest fluorescence. The DNaseAlert concentration was not doubled in the positive control by mistake.

Discussion

We might be comparing fluorescence signals which are quite low. Maybe we should once again increase the DNaseAlert concentration.

Saturday 04/08/18

Trial 4 (50 nM Cas12a, 62.5 nM crRNA, 200 nM DNaseAlert)

We followed the protocol with the same modifications as in the 3rd trial (200 nM DNase alert). This time, we also repeated the dilutions in order to obtain activator at concentrations of 1μM, 100nM, 10nM and 1nM (i.e. we tested samples of 100nM, 10nM, 1nM and 0.1nM concentrations). However we forgot to add the buffer after the incubation step.

Results

Analysis

It is clear that we couldn't activate the enzyme this time, since the fluorescence of all our samples is ranging near the negative control’s one at all time. However, we got an increase of fluorescence regarding our positive control compared to other trials (nearly two times more), which is consistent with the fact that we doubled the amount of DNaseAlert substrate, and indicates that we could have forgotten to do so in trial 3.

Discussion

Overall, we can conclude that this trial is a complete failure. This big difference in terms of fluorescent signal with the 3rd trial (same concentration of DNaseAlert substrate) was either due to the fact that we forgot to add the binding buffer in an adequate amount, or maybe because our crRNA has degraded in the meantime

Saturday, 11/08/18

Trial 5 (50 nM Cas12a, 62.5 nM crRNA, 160 nM DNaseAlert)

Another trial following the same protocol using Binding buffer protocol.

Results

Analysis/Discussion

The results are not good at all. Again, the negative control sample exhibits an abnormally high fluorescence, 100 nM sample is at the same level of fluorescence than the 1 μM

Trial 6 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert)

Another trial following the same protocol using Binding buffer, where we increased the concentration of LbCas12a to 75 nM and crRNA to 90 nM, compared to the previous trial.

Results

Analysis

First, the signal of 1 μM of activator was below the others, but it increased slightly above the 100 nM, which we were expecting at the end. Also the negative control is increasing which is maybe due to contamination of DNase I enzyme.

Discussion

This one is has shown the best results so far. At the end of the assay, the fluorescence signal reached the expected pattern.

mONDAY, 13/08/18

Trial 7

In this trial, we wanted to evaluate whether the increase in the concentration of Cas12 can give us higher level of signal. We performed this trial with two different concentrations of the enzyme, following the same protocol as usual (with Binding buffer).

Results

• (200 nM LbCas12a, 120 nM crRNA, 160 nM DNaseAlert)

• (100 nM LbCas12a, 62.5 crRNA, 160 nM DNaseAlert)

Analysis

We can observe an increase in negative controls overall.

Discussion

The problem could be due to contamination of samples with pipettes (because we used the same pipette for taking Dnase I for positive control). We're planning on repeating another trial using new pipettes tips and cleaning well our pipettes. Also, one of our TAs highlighted that Cas13a is very unstable at low salt, which means it might bind things that it is not meant to. It could be interesting to investigate the right concentration of buffer to use, since diluting it too much could lead to a very low sodium concentration which makes the enzyme promiscuous. The promiscuous binding could explain why our negative control actually cleaves.

Trial 8 (200 nM LbCas12a, 120 nM crRNA, 160 nM DNaseAlert)

We're still trying to evaluate whether the increase in the concentration of Cas12a can give us higher level of signal. We performed this trial with two different concentrations of the enzyme. This time we cleaned our pipettes carefully, used new sterile pipette tips, and made sure for every other aspect so that we wouldn't get any contamination.

Results

Error bars were drawn just for 1 μM, for more clarity.

Analysis/Discussion

This time the rise in sample fluorescence was more satisfying (the 1 uM almost reached the same level of activation as previous positive controls). However, we still have a very sharp rise for the negative control (with Cas12+crRNA), which made this trial again unsuccessful. However, the negative control without Cas12 was not activated at all, and even the level of signal decreased somehow.

Tuesday, 14/08/18

Trial 9 (200 nM LbCas12a, 120 nM crRNA, 180 nM DNaseAlert)

We Tried different concentration of salt to see if it influences the way the Cas12a system works. To do this we increased the binding buffer concentration as well as the NEBuffer 2.1 for another tube. We wanted to see which of the buffer worked better.

Results

• Activator as template

• Negative control as template

Analysis

The results are not good at all. Increasing the concentration of one or the other of the two buffers results in an even lower activation of the enzyme. Indeed, increasing the concentration of binding buffer reduces both signals in negative control and sample with activators. Also Cas12a doesn't get fully activated neither in 1X NEBuffer, nor in 5X, which suggests that using binding buffer in the future could be more relevant.

Discussion

We're not sure that using 5x of each buffer is the most efficient way of increasing the salt concentration within the assay, but it doesn't seem to fix our problem anyways, since increasing the concentration of buffer reduces both signals in negative control and sample with activators. However, we got convinced that using binding buffer is a better option, since it has shown a better activation of the Cas12a enzyme. We decided to use it for all the other trials to come.

Wednesday, 15/08/18

Trial 10 (200 nM LbCas12a, 120 nM crRNA, 180 nM DNaseAlert)

Compared with the previous trial, we have once again diluted our activators. In addition, we have transcribed and purified our crRNA again and this time we made sure that we did every step in the right way. We wanted to try different kinds of negative in order to check whether we had contamination in our of our tubes which could explain our high negative control.

Results

Analysis

The negative control sample which contains crRNA without Cas12a, does not produce a signal, which suggests that our purified crRNA is free of DNases.

Discussion

There is no Dnase contamination in our crRNA solution, nor in our Cas12 solution or in our DnaseAlert substrate. The activation of cas12a in our sample seems to be due to something other than contamination...

Friday, 17/08/18

Trial 11

In this experiment with tried different concentration of crRNA while keeping Cas12a concentration constant (50nM cas12a vs. 62.5nM/50nM/36.25nM/25nM of crRNA). We did the pre-incubation at 37°C for around 15minutes.

Results

• (50 nM LbCas12a, 36.25 nM crRNA, 200 nM DNaseAlert)

• (50 nM LbCas12a, 50 nM crRNA, 200 nM DNaseAlert)

• (50 nM LbCas12a, 62.5 nM crRNA, 200 nM DNaseAlert)

• (50 nM LbCas12a, 25 nM crRNA, 160 nM DNaseAlert)

Analysis

Once again the negative controls do not work and the fluorescent signal produced was above all the others which still makes no sense since the enzyme should not be active in that sample (no target in the solution).

Discussion

We were thinking of doing the experiments as in the NEB protocol given with the cas12a (with NEBuffer + 10min preincubation at room temperature and a ratio of 10:10:1 of cas12a/crRNA/activator). Also thinking of ordering the Ascas12a from IDT.

Sunday, 19/08/18

Trial 12

We repeated once again the assay, this time according to the Neb protocol given with LbCas12a

• 10 minute preincubation at 25°C
• same concentration of crRNA as cas12a

Results

Analysis

This method does not seem to resolve our negative control problem in which there is still a mysterious activation of the enzyme that is once again cleaving the ssDNA reporters without activation.

Discussion

We're going to stick to the other protocols based on our reference paper for the next trials...

Monday, 20/08/18

Trial 13

We're trying to test some combinations of different concentrations of LbCas12a and crRNA (while keeping the concentration of activator constant : 100 nM), to see which ones work the best for our system. Also testing whether not incubating the samples could lead to better results. This time, we didn’t first create a Master Mix with 4X concentration of Cas12+crRNA (200 nM of Cas12 + 250 nM of crRNA) as in the paper, where we needed to dilute them to 1X (50 nM of Cas12 + 62.5 nM of crRNA). Instead, we directly put the right amount in order to obtain the final concentration of Cas12+crRNA in each sample separately, and performed the assay with or w/o pre-incubation of the Cas12a/crRNA complex (for assembling).

Results

• FQ1 (50 nM LbCas12a, 62.5 nM crRNA, 160 nM DNaseAlert), with Pre-incubation

• FQ2 (75 nM LbCas12a, 62.5 nM crRNA, 160 nM DNaseAlert), with pre-incubation.

• FQ3 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert), with pre-incubation.

• FQ4 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert), without pre-incubation.

Analysis

We were able to get good activation for each of our samples (FQ1, FQ2, FQ3 and FQ4) with a relatively low rate of activation in the negative control for each except for the second one. Surprisingly, the experiment without pre-incubation gave up the best yield in terms of fluorescence.

Apart from that, we can also see through the error bars that we have a lot of variations between our two measured duplicates (not drawn for negative and positive controls), and from this, the only sample to consider for success would be the FQ4 one (without pre-incubation).

Discussion

We finally concluded that the problem was related to the Master mix step: the cas12a and crRNA are incubated at too high of a concentration. Also, in the samples without pre-incubation we were able to get a very good result which could mean that pre-incubation made Cas12a not as activated as it should be.

Besides, we're getting too much variation for each sample. Should we use more replica for each sample (right now we are using two replicas) ? or is it from the device ? Another possible sources of errors would be either pipetting or bubbles formation...

Tuesday, 21/08/18

Trial 14

Following the same hypothesis of the 13th trial, we're still looking for the ideal concentrations of LbCas12a and crRNA to use in our assay. Four Samples (100 nM of dsDNA activator) where either left to pre-incubate or not, and contained different amounts of the enzyme and crRNA. No master mix was prepared. We also did the pipetting more carefully and tried our best to avoid bubbles.

Results

• FQ1 (50 nM LbCas12a, 62.5 nM crRNA, 160 nM DNaseAlert), with pre-incubation

• FQ2 (75 nM LbCas12a, 62.5 nM crRNA, 160 nM DNaseAlert), with pre-incubation

• FQ3 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert), with pre-incubation

• FQ4 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert), without pre-incubation

Analysis

We can see the effect of pre-incubation here: without pre-incubation the slope is lower than with pre-incubation for the same concentrations of both Cas12a and crRNA (FQ3 vs FQ4). Also, we can see that the 75 nM of Cas12a and 90 nM of crRNA are the most efficient concentrations here (regardless of the incubation) since these samples (FQ3 and FQ4) gave out the highest signal.

Discussion

It looks like we're going in the right direction for figuring out the optimal concentrations to use in our assay.

Friday, 24/08/18

Trial 16

We did this experiment with the same hypothesis as in the previous two trials. We ran different assays with different concentrations of Cas12a and crRNA. All samples were incubated (for Cas12a assembling).

• FQ6 (62.5 nM LbCas12a, 75 nM crRNA, 160 nM DNaseAlert), with pre-incubation

• FQ2 (75 nM LbCas12a, 62.5 nM crRNA, 160 nM DNaseAlert), with pre-incubation

• FQ3 (75 nM LbCas12a, 90 nM crRNA, 160 nM DNaseAlert), with pre-incubation

Analysis

Overall, this Trial was a success, and by looking at the slopes, we can say that the optimal concentrations of Cas12a and crRNA for our system were obtained for FQ6 sample (62.5 nM LbCas12a and 75 nM crRNA), since we got the sharpest slope, i.e. faster activation of Cas, for our sample of interest, which surpassed the positive control.

Discussion

We finished our optimization of the concentrations after obtaining a very nice result with our FQ6 sample, and an activation which reaches its maximum after approximatively 80 minutes. For the next assays, we're planning on working with 62.5 nM of LbCas12a and 75 nM crRNA while maybe trying to change the concentration of dsDNA activator to see how it affects the signal.