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.

Notebook Chromosomal rearrangements

Wednesday, 05/09/18

PCR amplification of Bcr-Abl junction

In the PCR amplification of Bcr-Abl junction series of experiments, we want to amplify the Bcr-Abl junction in order to detect it further with LbCas12a. We're going to start from extremely low concentrations of template (1 pM and/or 10 fM) and try to amplify them with different primers (three forward primers and two reverse primers), each containing an inserted PAM sequence. There are two main aims:

• Overcome the fact that there might exist no suitable PAM sequence (TTTN for Cas12a) nearby the SNP site or junction, for the Cas12a recognition assay.
• Prove that these fragments can be specifically detected in very small quantities by combining the sensitivity of PCR amplification method and the specificity of CRISPR-Cas system, this in order to get as close as possible to the concentrations present in the blood, and so be able to establish a proof of concept about the possibility of such detection in blood plasma.

Please reffer to the PCR using Phusion® High-Fidelity DNA Polymerase protocol.

Approach: We ordered the sequence corresponding to the junction as gBlocks from Integrated DNA Technology (IDT), as well as several base pairs of the two normal genes (Bcr and Abl) (Fig.2, 3) that extend on both sides of the junction. Primer oligos were ordered from IDT (c.f. Fig.1):

Forward primers (from top to bottom, Fig.1 below):

• F_primer_chromo_symu_01 (F1)
• F_primer_chromo_symu_04 (F4)
• F_primer_chromo_symu_03 (F3)

Reverse primers (from left to right, Fig.1 below):

• R_primer_chromo_symu_02 (R2)
• R_primer_chromo_symu_01 (R1)

Resuspension of the gBlocks/Oligos was done as following, according to this protocol:

Template DNA solutions (dilutions) were prepared as below:

• Bcr-Abl (BA-)
• Bcr (BC-)
• Abl (AB-)

Then, the 10 µM dilutions of the primers (for PCR use) were prepared:

Hypothesis:

• Bcr-Abl DNA sequence amplification should result in an amplicon of 105-109 bp when using R2 primer and either of the forward ones, or 142-146 bp if using R1 primer.
• Amplification of Bcr and Abl fragments is linear (i.e. only forward primer annealing on Bcr template, reverse on Abl) rather than exponential, meaning that we shouldn't be able to observe it on the gel, given the low initial concentration (1 pM or 10 fM). Also, amplification of Bcr fragment should result in an amplicon of ~239 bp (F1 primer), while for Abl, the amplicon obtained with R1 is of 316 bp length, and 279 for R2 (see Fig.4):

Cas12a detection assays

In this series of experiments, we want to be able to detect specifically the presence of the junction (previously amplified by PCR), using Cas12a system. Recognition of the activator by the crRNA unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that will completely degrades our ssDNA reporter molecules (DNaseAlert, IDT), which results in a higher fluorescent signal. For the CRISPR RNA design (crRNA), we chose to use a shorter guide sequence (17 bp rather than 20), designed to recognize the sequence right after the PAM introduced by primer F1, F3 but not F4. First, the choice of using a shorter crRNA is based on the work done by Li S, Cheng Q, Wang J et al.1, where they proved that point mutations within a large region (1st–16th bases) resulted in more than 2-fold difference in fluorescence signals for both 16-nt and 17-nt crRNA guide sequences.

We followed the Fluorophore-Quencher reporter Cas12a assay. crRNAs oligo fragments were annealed with a T7 primer and transcribed then purified following respectively the crRNA Transcription using T7 RNA Polymerase (Promega) and crRNA purification (ZYMO Research RNA Clean & Concentrator™-5 Kit) protocols.

Hypothesis:

• Cas12a enzyme should be activated by two of the amplified templates (Bcr and Bcr-Abl junction); however, we should detect a signal that is 2-fold less (at least) intense when using the separate gene (Bcr) as activator, since it's target strand (TS, not shown here for clarity) present several mismatches with the used crRNA, which is identical to the non-target strand (NTS) (see Fig.5).
• Also, Abl amplified fragment should not activate the Cas12a system, since it's amplified only with one reverse primer that does not contain the PAM sequence.
• Moreover, fragments amplified with the forward primer four (F4) should not activate the Cas neither, since the PAM sequence is shifted from a base.

PCR amplification of Bcr-Abl junction-Trial 1

In this version, we're performing the assay with one forward primer only (F_primer_Chromo_symu_01), the second reverse primer (R_primer_chromo_symu_02)and different annealing temperatures for the PCR reactions, i.e. three duplicates of the two samples (A-A1), in order to select the right temperature to use for the amplification, since it was difficult to estimate the melting temperature of the primers due to the mismatches with the template sequence.

• 59 °C : 1st duplicate.
• 64 °C: 2nd duplicate.
• 69°C: 3rd duplicate.

The thermocycler was programmed as described below. IMPORTANT: three blocs were used for proceding with the samples simultaneously, since we have three sets with different forward primers.

As controls, we prepared a PCR negative control sample in which we only add water and no template (D), and two others (N and N1) that contain the initial concentrations of template without amplification (in order to be sure that we can't detect these without amplification). Gel was load as following:

Results

Analysis/Discussion

Overall, an amplification was obtained for all annealing temperatures tested, and we were able to detect the expected amplicon of 105 bp. Moreover, we couldn't observe any band in the negative control's lane, which means that our samples were free from any DNA contamination. We can however argue that we got a better amplification when the annealing step was performed under 69°C.

We can state that the most efficient amplification was achieved when we annealed our primers (forward F1 and reverse R2) at 69°C. Since it was the one found using the Neb calculator (that doesn't take into account mismatches though), we decided to trust it for our further amplifications.

References

1.Li, S., Cheng, Q., Wang, J., Zhao, G. & Wang, J. CRISPR-Cas12a-assisted nucleic acid detection. 5,1–8

Thursday, 06/09/18

PCR amplification of Bcr-Abl junction-Trial 2.

In this version, we performed the assay with our three different forward primers (TTTN PAM sequence introduced with two mutations near a thymine, different lengths, GC content, annealing temperatures) and R_primer_chromo_symu_02 (R2) reverse primer, to see which one works the best (in terms of amplification). The annealing temperatures for each set of primers (one reverse primer used), was calculated using NEB Tm calculator. As controls, we prepared a PCR negative control sample in which we only add water and no template, and two others that contain the initial concentrations of template without amplification (in order to be sure that we can't detect these without amplification).

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

• PCR samples:

Amounts (ul)	A (1pM template)	A1 (10 pM template)	B (1 pM template)	B1 (10 fM template)	C (1 pM template)	C1 (10 fM template)	D: (-) PCR control	Master Mix	Final concentrations
Nuclease-free water	28.5	28.5	28.5	28.5	28.5	28.5	28.5	270.75	-
5X Phusion HF buffer	10	10	10	10	10	10	10	95	1X
10 mM dNTPs	1	1	1	1	1	1	1	9.5	200 µM
From Master mix	39.5	39.5	39.5	39.5	39.5	39.5	39.5	375.25	-
10 µM F1-10	2.5	2.5	-	-	-	-	2.5	-	0.5 µM
10 µM F3-10	-	-	2.5	2.5	-	-	-	-	0.5 µM
10 µM F4-10	-	-	-	-	2.5	2.5	-	-	0.5 µM
10 µM R2-10	2.5	2.5	2.5	2.5	2.5	2.5	2.5	-	0.5 µM
Nuclease-free water	-	-	-	-	-	-	5	-	-
100 fM Bcr-Abl Template (BA-4)	-	5	-	5	-	5	-	-	10 fM
10 pM Bcr-Abl Template (BA-2)	5	-	5	-	5	-	-	-	1 pM
Phusion DNA Polymerase	0.5	0.5	0.5	0.5	0.5	0.5	0.5	-	1.0 units/50 µl PCR
Total volume	50	50	50	50	50	50	50	-	-

• The thermocycler was programmed as described below. IMPORTANT: three blocs were used for proceding with the samples simultaneously, since we have three sets with different forward primers.

98 °C	30 sec (Initial Denaturation)
98 °C	10 sec (denaturation)	35 cycles
A-A1-D (F1/R2): 69°C.                            B-B1 (F3/R2): 72°C.                               C-C1 (F4/R2): 72°C.	30 sec (primer annealing)	35 cycles
72 °C	30 sec (extension)	35 cycles
72 °C	10 min (Final Extension)
4 °C

Results:

Analysis

We could observe a unique band of roughly 100 bp for each tested sample, which would in principle say that the amplification worked well, since we're expecting amplicons of 105 bp (F1 and F4 primers) and 109 bp (F3). However, we got the same band in our negative control sample (water, no DNA template), in which we sould not observe anything, and this suggests a possible DNA contamination of our PCR reaction samples. We could also notice the absence of any band for the 1 pM/10 fm (without amplification) samples, which is coherent with the fact that we can't detect such low concentrations of DNA without amplification.

Discussion

Can we really conclude as to the success of this pcr knowing that our negative control did not work ? Anyway, we're going to do the same PCR again in order to be sure.

Friday, 07/09

PCR amplification of Bcr-Abl junction-Trial 3.

Same approach as in the previous trial. This time, we prepared samples containing the Bcr and Abl genes as templates (1 pM), to compare the efficiency of amplification and assess the linear amplification results (see "hypothesis", point two). We amplified them using F_primer_chromo_symu_01 (F1) primer and R_primer_chromo_symu_02 (R2).

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Analysis

Same results as trial 2 unfortunately, we got again this band in our negative control sample. Also, we can observe the exact same band in the samples containing the Abl and Bcr coding sequences, respectively. This suggests that we got an exponential amplification for these rather than linear one. This was not expected, since only one of the primers used is complementary to each sequence (respectively F1 with Bcr and R2 with Abl). Also, the amplification of these two sequences should've resulted in amplicons with respectively 239 and 279 bp (c.f. the hypothesis of the experiment, listed on the 05/09), while in this scheme it seems that the same template was amplified...

Discussion

Since it's the second time that we obtain this strange result for the negative control, and the band is the exact replicate of the one obtained in all the other samples, this excludes the hypothesis of a random DNA contamination. We can state that either we put (again) one of the DNA templates (Bcr-Abl junction most probably) in the control by mistake, or one of the 10 µM primer solution is contaminated with DNA template. As for the Bcr and Abl samples, this same band doesn't make sense at all...

Sunday, 09/09/18

Another trial with the same hypothesis as the previous one (amplification done with reverse primer two i.e. R2 for all samples). Still trying to figure out the origin of the band we got in our negative control sample, and to obtain the right amplification for each DNA template (i.e. three different amplicons).

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Analysis

We still have our strange contamination for the negative control sample. Same pattern of bands.

Discussion

It looks like we're obtaining the same amplicon in each sample, while two of them contain different DNA templates. This could suggest a contamination in one of our 10 µM primer stocks that we've been using so far, perhaps the forward primer one (F1), used to amplify different DNA templates, or the reverse primer two (R2), the only reverse primer used so far.

Monday, 10/09/18

Cas12a detection assay-Trial 1

We performed our first Cas12a assay for detecting the junction, using the PCR products obtained during "PCR amplification of Bcr-Abl junction-Trial 4", following the hypothesis elaborated on the 05/09.

Results

• Cas12a assay using different amplicons (as activators) obtained from the amplification of the Bcr-Abl junction with different forward primers (and R2 as reverse primer).



• Cas12a assay performed with F1-R2 amplicons (activators) obtained from both the junction and Bcr-Abl genes.

Analysis

Several observations can be made:

• We can clearly see that the primer pair F1-R2 gives the best results (Bcr-Abl junction amplification), followed by F3-R2 and F4-R2. Indeed, the F1-R2 amplicon obtained from the amplification of 1 pM junction part significantly activates the Cas12a enzyme and generates a signal ~1.5 times as strong as the one obtained using Bcr or Abl separate genes. Regarding Bcr, this fits more or less with our hypothesis, however Abl gene shouldn't activate the Cas at all.
• Several amplicons of the junction part (F1-R2, F3-R2 both for a concentration of 10 fM and 1 pM before amplification, see first graph) yield almost the same signal than Bcr and Abl normal genes. This was not expected, due to the point mutations present in the target strand recognized by the crRNA, and that should have resulted in much less activation of the Cas12a system, with no activation with Abl.
• Activation with F4-R2 amplicon as a target, which shouldn’t be recognized by our crRNA.

Discussion

Desired results obtained just with the F1-R2 primers amplification of the junction: ~1.5 folds decrease in signal when using Bcr or Abl as activators in comparison. We have to figure out why our Cas12a enzyme is being activated with F4 amplicons and Abl..

Tuesday, 11/09/18

PCR for negative controls without any DNA

We wanted to understand why our negative control showed some amplification. The aim is to see whether some of our samples are contaminated. For this, we created six PCR (-) control samples, in which we only added water and different combination of primers (see below). Afterwards, we ran a gel with two extra samples: a lane containing Phusion enzyme alone, and one with loading dye, in order to verify that these samples are indeed free of contaminations.

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Analysis

It is hard to really know what do the bands actually represent but one of our suspicion is that our reverse primer 2 stock (R_primer_chromo_symu_02) is contaminated by one of our template (probably the Bcr-Abl junction since we got the desired band of ~ 105 bp) and that the bands when the reverse primer 1 was used somehow represent the primers (hetero-dimers ?)

Discussion

Since we performed the dilutions of our primer stocks again (resuspended at 100 µM) in order to obtain the 10 µM primer solutions that we can use for our PCR, we are convinced that the reverse primer 2 (R2) stock was somehow contaminated with Bcr-Abl junction.

Wednesday, 12/09/18

PCR amplification of Bcr-Abl junction-Trial 5

Our hypothesis was that our reverse primer 2 stock solution (R_primer_chromo_symu_02-R2) is contaminated. In this trial we tried amplification with our reverse primer 1 (R_primer_chromo_symu_01-R1) to see if it would differ from the amplicon produced using the contaminated one (R2). We used 1 pM DNA template for each sample (either Bcr-Abl, Bcr or Abl alone), and two negative controls (no DNA) containing different combinations of primers.

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Analysis

The amplification seem to have worked using R1 (~145 bp).

Discussion

We can quite confidently say that our R2 reverse primer was contaminated. We'll perform a Cas12a assay with these PCR products.

Cas12a detection assay-Trial 2

We performed another Cas12a assay, using the PCR products obtained from the PCR above ("PCR amplification of Bcr-Abl junction-Trial 5").

Read notes of 05/09 for approach and hypothesis for the general "Cas12a detection assays" set of experiments.

Reults:

Analysis/Discussion

Obviously this trial did not work. What could have happened:

• Problem with Cas12a : crRNA might have been degraded
• Amplification of the wrong template

Since the positive control (DNase I sample) is working fine, we can be confident that the DNaseAlert wasn't degraded.

Thursday, 13/09/18

PCR amplification of Bcr-Abl junction-Trial 6

We performed one more time the PCR on our templates (1 pM concentration before amplification), this time using only forward primer 1 (F1) and one of the two reverse primers (R1 or R2). Negative controls included F1 primer and either one of the reverse primers, amplified with water as template. As for the gel, we also ran different samples containing F1 forward primer, R1 and R2 reverse primers (diluted at 50 µM, without amplification), as well as two samples containing annealed primers F1-R1 and F2-R2 (50 µM, without amplification), in order to figure out whether the bands we were obtaining so far were due to dimers.

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Analysis

• The junction (Bcr-Abl sample) was not amplified by the reverse primer 1, but amplified successfully with the second reverse primer. Is it some manipulation error ?
• Bcr seems to be amplified with both combinations of primers (F1-R1 and F1-R2), However, the size still do not make sense (size between 75 and 200 bp rather than the 239 expected). Abl was not amplified with F1-R1, compared to the discrete band obtained when F1-R2 were used. According to our hypothesis (c.f. wednesday 05/09), we shouldn't be able to observe the PCR products of these two samples on a gel due to the lack of sensitivity of the SYBR Safe nucleic acid stain, given that the concentration of the amplification products is expected to be really low because of the linear amplification (rather than exponential) that is occuring in these samples (~35 pM since we started from 1 pM of DNA concentration).. The bands obtained for Abl could be due to contamination (i.e. amplification with Bcr-Abl contaminated primer R2); however, for Bcr F1-R1, it doesn't really make sense.
• As for the control sample in which we added water and F1-R2 primers, the amplification resulted in an amplicon of the same size as we would expect if we had amplified the Bcr-Abl junction, thus suggesting that our reverse primer 2 is indeed contaminated. Also, adding this primer without amplifying it resulted in a completely different band, suggesting that the amplification is essential in order to obtain a quantity such that we can observe Bcr-Abl fragments on gel.

Discussion

the only deduction that can be drawn from this gel is that our concentrated 100 µM stock of the reverse primer 2 (R2) is contaminated. From now on, we decided to work with the first reverse primer (R1).

Friday, 14/09

Cas12a detection assay-Trial 3

We performed another Cas12a assay, using the PCR products obtained from the PCR above ("PCR amplification of Bcr-Abl junction-Trial 6").

Read notes of 05/09 for approach and hypothesis for the general "Cas12a detection assays" set of experiments.

Results

Analysis

• Bcr-Abl F1-R1 amplicon (amplified part of the junction containing the PAM sequence) is up to four times less activating the Cas12a enzyme compared to the other gene templates used (Bcr and Abl amplicons, respectively), which is the contrary of what we're expecting to see. Moreover, we do not expect Abl to be an activator for the Cas12a (no PAM sequence).
• Bcr-Abl F1-R2 induces a much higher signal but still way less than Bcr and Abl separate genes.
• Both PCR negative control samples ("Water_F1-R1/F1-R2") are surprinsigly activating the Cas12a enzyme, resulting in a fluorescent signal which is clearly unexpected, since these samples contain no DNA template before their amplification...
• Bcr-Abl without amplification (no PAM sequence) sample (dashed purple line) did not activate the enzyme, which confirms that the absence of the PAM sequence is clearly preventing the Cas12a from cleaving the dsDNA template (Bcr-Abl fragment), thus preventing the trans-cleavage of DNaseAlert reporter ssDNA fragments.

Discussion

Overall, this trial is a disaster. We did not reach the corresponding levels of activation that we're expecting for the different dsDNA activators (c.f. the hypothesis in "Cas12a detection assays", drawn on the 05/09). More strangely is that our dsDNA free samples are somehow activating the Cas12a enzyme. Is our R1 primer also contaminated ? or is it F1 unstead ?? How about Abl being an activator ? Anyway, we're going to perform another assay to figure out.

Saturday, 15/09/18

PCR amplification of Bcr-Abl junction-Trial 7

We performed one more time the PCR on our templates (1 pM concentration before amplification), this time using either forward primer 1 (F1) or forward primer 4 (F4), and reverse primer 1 (R1). Negative control included F1 primer and R1 reverse primer in combination with water.

Here, we also wanted to test the specificity of the PAM-Cas12a interaction. For this, we amplified both our Bcr-Abl junction and Bcr gene with forward primer 4 (F4), with the hypothesis being that the resulting amplified products shouldn't be recognized by Cas12a, since the crRNA used is not complementary to the sequence right after the PAM sequence added by this primer. Also for this trial, 30 amplification cycles were ran instead of 35 used until now for the PCR reaction with PHUSION polymerase (Thermo Fisher scientific).

Read notes of 05/09 for approach and hypothesis for the general "PCR amplification of Bcr-Abl junction" assay.

Results

Sunday, 16/09/18

Cas12a detection assay-Trial 4

We performed another Cas12a assay, using the PCR products obtained from the experiment "PCR amplification of Bcr-Abl junction-Trial 7" done during the previous day. Read notes of 05/09 for approach and hypothesis for the general "Cas12a detection assays" set of experiments

Results

Analysis

• Roughly the same observations as in the previous Cas12a detection assay.
• Activation of Cas12a with the Bcr-Abl F4-R1 amplicon. This is indeed in disagreement with our hypothesis.
• High signal obtained for the PCR negative control sample ("Water_F1-R1", dashed blue line).

Discussion

We're starting to think that the dsDNA contamination that we're facing so far may be due to primer F1. The presence of some Bcr-Abl template DNA in this primer would explain why we're getting such an activation with our PCR products containing Bcr or Abl, as well as the negative control result. We decided to order F1 (F_primer_Chromo_symu_01) as well as R2 (R_primer_Chromo_symu_02) again from Integrated DNA Technologies (IDT).

Thursday, 20/09/18

Cas12a detection assay-Trial 5

Aim:

For this trial we wanted to check wether the primers somehow activated our cas12a by doing hetero dimers. In this way we could eliminate the possibility of dimers and prioritize the hypothesis of contamination.

The protocol we followed is : FQ reporter assay V.5 -For primers [20.09.18]

Read notes of 05/09 for approach and hypothesis for the general "Cas12a detection assays" set of experiments

Results:

Discussion:

We can see that the annealed primers as well as the individual primers do not activate cas12a. The samples were not amplified which mean that our primers can be contaminated.

Friday, 21/09/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 1

Aim:

To see whether our usual PCR worked correctly when done in plasma.

Results:

Wednesday, 26/09/18

PCR amplification of Bcr-Abl junction (20 cycles)-Trial 8

Aim:

Amplification of the Bcr with the newly received F1 and R2 primers as our old one seemed to have been contaminated.

Results:

Discussion:

Seems to work! Only the Bcr-Abl has been amplified as should be. But contamination (on the comb? in the ladder?)

Thursday, 27/09/18

Aim:

To try the gel again with the previous pcr products (20 cycles) as the last gel was contaminated

Results:

Discussion:

The amplification seems to have been successful! Only the bcr-abl sequence seem to have been amplify. The bands are quite faint so we will reproduce the same PCR with more cycles.

Cas12a detection assay-Trial 5 (from PCR products trial 9)

Discussion

The results are not as expected: the negative control N of the cas12a assay (Cas12a with crRNA and DNaseAlert) is highly fluorescent. The sample in which we would like to detect the highest level of fluorescence, Bcr-Abl with F1-R2, is quite low compared to samples which should barely activate the enzyme (Bcr F1-R2).

Friday, 28/09/18

PCR amplification of Bcr-Abl junction (30 cycles)-Trial 10

Results:

Discussion:

As we can see the results differ a lot from the previous gel (20 cycles). There seem to be some contamination althought the R1 and F1 primers are new. To make sure it is contamination that happened between this gel and the last we will reproduce those same pcr.

Saturday, 29/09/18

PCR amplification of Bcr-Abl junction (20 cycles)-Trial 11

Aim:

To remake the same pcr as previous to check for contamination

Results:

Discussion:

The gel is the same as the first 20 cycles gel. This suggest that there was no contamination in the F1, R2 primers or nuclease free water between the previous 20 cycles gel and the 30 cycles gel. We want to see the 30 cycles amplification again.

Cas12a detection assay-Trial 6 (from PCR products trial 11)

Discussion:

The negative control is not as high as it previously was but still too high. We think there might be some problem with the new Cas12a protein we received. The Bcr-Abl F1-R2 is the sample which activates cas the most which is what we want! But the Abl F1-R2 sample shows the highest fluorescence which is not a good thing since abl sequence is not only not complementary to the crRNA but also doesn't contain the PAM sequence.

Sunday, 30/09/18

PCR amplification of Bcr-Abl junction (30 cycles)-Trial 12

Aim:

To remake the 30 cycles pcr in order to see how sensitive and specific our amplifcation is.

Results:

Discussion:

Again there are some bands in the negative controls as well as in the bcr and abl lanes. Next time we will either put a lower concentration to start with or/and put background DNA (from salmon sperm DNA). Hopefully the negative controls will be empty again.

Cas12a detection assay-Trial 7 (from PCR products trial 12)

Discussion:

The negative control is once again way too high. We will try to do it in plasma next time as we think that maybe having background DNA will make the amplification more specific.

Monday, 01/10/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 13

We wanted to try amplificaiton in Plasma again as we believe that having some sorts of DNA background could help our amplification specificity. We chose to make a 30 cycles reactions to have clear bands.

Results:

Cas12a detection assay in plasma-Trial 1 (from plasma PCR products trial 13)

Results:

Analysis:

The activation is overall very small. In this assay we used the remaining of an older cas12a protein. Maybe the concentration of the protein wasn't high enough. Another possibility is that the amplification was not successful.

Wednesday, 03/10/18

PCR amplification of Bcr-Abl junction with Plasma (30 cycles)- Trial 14

As last cas12a assay revealed no activation we decided to try again with the same samples. We removed the primer F4 as we don't find it necessary.

The bands look very faint for a 30cycles amplification.

Cas12a detection assay in plasma-Trial 2 (from plasma PCR products trial 14)

Hooray! This assay is sucessful!! Bcr-Abl is activated as it should be. The other samples show small to no activation at all.

Thursday, 04/10/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 15

Here we wanted to try different kind of Bcr-Abl concentration to see how the cas12a activation would change.

Cas12a detection assay in plasma-Trial 3 (from plasma PCR products trial 15)

Friday, 05/10/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 16 (25 cycles)

We decided to change our approach and to look at the difference in activation of a sample with healthy cfDNA (the background: Bcr and Abl) compared to a sample which contains both the background and the mutated cfDNA (Bcr-Abl). This is indeed closer to the reality as a patient with cancer will have both ctDNA and cfDNA corresponding to the healthy cells.

There is very faint bands on the gel which is maybe due to the fact that we only did around 25 cycles.

Cas12a detection assay in plasma-Trial 4 (from plasma PCR products trial 16)

Sadly the results are not as expected.. The background which contains no Bcr-Abl activated the Cas12a targeting Bcr-Abl more than the sample that actually has template.

Tuesday, 09/10/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 17 (30 cycles)

Today we wanted to reproduce the same kind of experience as yesterday but with two different concentrations as we've previously seen that template concentrations can sometimes affect cas12a.

Cas12a detection assay in plasma-Trial 5 (from plasma PCR products trial 17)

Samples 1 :

• b1 1pM
• BAb 10fM

Samples 2:

• b2 100fM
• BAb2 1fM

Again, a very high negative control from plasma. It is not surprising as there was a band in the gel above for the negative control. Except this we can see that the samples containing the targeted Bcr-Abl junction is in both case more activated than the ones without (the background). This is a good thing. Next time we'll try the same again with possibly different concentrations and hopefully we'll get a sucessful negative control.

Wednesday 10/10/18

PCR amplification of Bcr-Abl junction with Plasma - Trial 18 (30 cycles)

We tried to do the amplification one last time under the same conditions as last time. In addition we tried another concentration.

We can see that the samples containing the Bcr-Abl segment are brighter and larger than the one without. The problem is that even the negative control has a band: the same band which are present in every background only sample.

Cas12a detection assay in plasma-Trial 6(from plasma PCR products trial 18)