Team:Freiburg/Project/method

uniBAss - uniCAS Binding Assay

uniBAss - The innovative & easy screen for binding affinity

We developed a novel ELISA-based method to assess the binding efficiency of our proteins: the uniCAS Binding Assay uniBAss. It is a powerful tool for the biochemical characterization of the binding capacity of our dCas9 fusion proteins and our truncated dCas9 versions with high throughput capabilities.

1. First a 96-well ELISA plate is coated with streptavidin. Then biotinylated oligonucleotides containing the DNA sequence complementary to the crRNA incorporated into dCas9 are applied.

2. 42h after transfection with the dCas9-fusion constructs that will be tested and its desired target site on the RNAimer plasmid HEK293T cells are taken up in dilution buffer and lysed by sonifying. Then the dCas9/crRNA containing cell lysate is ready to be applied to the wells.

3. The dCas9 in the cell lysate can now bind via its incorporated crRNA to the complementary oligo on the bottom of the well.

4. After a washing step the first antibody is added. This mouse anti-HA antibody binds to the HA-tag at the N-terminus of dCas9.

5. When the unbound first antibody is washed away the secondary antibody can be added. The secondary anti-mouse-antibody is coupled to a horseradish peroxidase (HRP) that catalyzes the oxidation of ABTS by hydrogen peroxide resulting in a change of color.

6. The absorption at 405 nm is then measured and represents the amount of dCas9 bound in each well. To substract out differences in dCas9 expression levels the uniBAss data can be normalized to western blot data to reflect binding properties of dCas9 to its target site.

Results

The uniCAS Binding Assay enables us to verify that our dCas9-fusion proteins show the same DNA binding capacity as dCas9 without an effector. Thus uniBAss can be used to quantify the amount of HA-tagged DNA binding dCas9 and therefore helps us to characterize our constructs more closely.

Figure 1: Binding affinity of dCas9 fusion constructs after normalizing the dCas9 amount with the help of a western blot

Mammalian HEK293T cells were transfected with dCas9, dCas9-VP16, dCas9-KRAB and dCas9-G9a and the RNAimer plasmid targeting EMX1. 42h post transfection the standard uniBAss protocol was followed. Constructs we analyzed in technical duplicates to assess the accuracy of uniBAss. For every construct the ABTS readout (background level substracted) was normalized to western blot dCas9 expression data obtained from the same cell lysates. Through this normalization we could compensate differences in protein expression between the constructs and thereby compare the actual binding properties. Figure 1 shows the normalized uniBAss data for dCas9, dCas9-VP16, dCas9-KRAB and dCas9-G9a + RNAimer(EMX1) when targeted to biotinylated EMX1 oligos. Transfection controls without dCas9 or the RNAimer plasmid did not show detectable signals in our binding assay.

Development of uniBAss

The need to characterize the behavior of CRISPR/Cas is motivated by several means. Different fusion constructs have been build based on the CRISPR/Cas system such as our dCas9-VP16 or dCas9–KRAB constructs, but it remained unclear if the binding characteristics of dCas9-VP16/KRAB are different from dCas9 alone. Simulating complex gene circuits in-silico generates new needs for straight forward characterization of the binding affinity followed by quantification. By this mean information can be gathered about a variety of promoters for different dCas9 fusion constructs. Different assays have been developed to characterize protein-DNA interplay to analyze their respective binding behavior [1]. Examples of these protein characterization assays are the Chromatin Immunoprecipitation (ChIP) and the DNA Electrophoretic Mobility Shift Assay (EMSA). Both systems have individual strength as well as limitations. ChIP can be used to quantify a sample when coupled with qPCR analysis. However ChIP is limited by the absence of high throughput possibilities [2]. EMSA is able to detect low abundance DNA binding proteins from lysate with a high sensitivity but it is difficult to quantitate a sample [3]. However, those assays do not address the need to analyze the binding of a protein to its respective DNA sequence and thus perform a quantification with high capabilities. Starting from these needs a new assay for the biochemical characterization of dCas9 had to be established with the requirements of binding capacity test and quantification with high throughput capabilities. Therefore we developed our Enzyme-linked Immunosorbent Assay (ELISA) uniBAss. The assay is based on a streptavidin-biotin interaction [4] with the 5’ biotin tag of the oligonucleotide [5] was used to immobilize the target DNA.

Optimization

To obtain robust conditions for uniBAss, several experiments were performed. Different salt concentrations as well as different oligonucleotide lengths were examined to optimize this assay. Hereafter different dCas9 fusion proteins were tested regarding their DNA binding capacity. In addition Cas9 versions with one nickase domain and without a nickase domain were compared to investigate the influence of the nicking enzyme towards the binding behavior. Subsequently to the optimization process the resulting buffer condition 10 mM Tris, 1 % BSA, 90 mM NaCl, 5 mM MgCl₂ containing EDTA free Protease inhibitor was used to test different dCas9 fusion constructs.

Figure 2: Binding restrain of the Cas9 MX1 crRNA complex for varying MgCl2conditions

Figure 2 and 3 show that the binding behaviour of the dCas9 / crRNA EMX1 strongly depends on the salt concentrations.

Figure 3: Binding restrain of the Cas9 MX1 crRNA complex for varying NaCl conditions

The experiment(figrue 3) showed that dCas9 with the EMX1 crRNA sequence acts differently for different buffers. Therefore an optimization process was performed focusing on two main points. Thus different salt concentrations illustrate one parameter for optimization. As another parameter the length of the oligonucleotides coated onto the ELISA plate was taken into account for characterization.

Figure 4: Design of the different oligos

Figure 5: DNA binding capacity tested with different oligo-lengths

Using different oligonucleotide lengths to determine the best size of the coated oligonucleotides revealed that the presence of a longer sequence at the 5’ end with about 11 nucleotides consisting of the crRNA locus is essential to get dCas9 to bind. The presence of a second long sequence at the 3’ end of the crRNA however is not essential for simple binding assays but enables a more robust binding environment. Biotinylated oligonucleotides for prospective assays were designed analogue to the longest oligonucleotides used in the attempt to offer a point of interaction for fusion proteins. The second parameter focused on the buffer conditions. The used buffer is composed of 10 mM Tris, 1 % BSA and varying concentrations of MgCl₂ and NaCl. The salt concentrations were varied against each other generating 16 different buffers.

Factorial design

The second part of the optimization was done using a factorial design. This approach was used to test whether the MgCl₂ concentration is linearly linked to the ABTS conversion. Therefore different concentrations of MgCl₂ – 30 mM, 20 mM, 10 mM and 5 mM – and of NaCl – 150 mM, 120 mM, 90 mM and 60 mM, were varied against each other. Indicating that for high MgCl₂ concentrations the binding of dCas9 is close to zero (Figure 6). Whereas with descending MgCl₂ concentrations more dCas9 is able to bind to the oligonucleotides, indicating an increase in an exponential matter. This is true for all the tested NaCl concentrations however it is to mention that 150 mM NaCl has the lowest bound dCas9.

Figure 6: Factorial design expriment to test the behaviour of the dCas9 towards different salt environments

The figure 6 shows that the binding behaviour of the dCas9 / crRNA towards EMX1 display similar behaviour for descending MgCl₂. This is true for all four NaCl concentrations however 150 mM NaCl has the smallest yield.

Use of factorial design

To test if the curves can be fitted to an exponential increase within decreasing MgCl₂ concentration the factorial design approach was performed to analyze the gathered data (Table 1). To check if the conditions either behave in a linear or a non-linear way the experimental b0 values were compared to the calculated b0 values resulting from a central point. The results of this analysis are shown in Table 1. It was oberserved that the amount of bound dCas9 increases with a reduction of the MgCl₂ concentration.

Table 1: Factorial design approach

The plotting of the data in two dimensions indicated that there is an exponential increase of the ABTS conversion with descending MgCl₂ concentrations. To verify this, a factorial design with different MgCl₂ concentrations was performed within a range of 30 mM and 5 mM MgCl₂. After having the data illustrated in three dimensions (figure 7), it occurred that the decreasing amounts of NaCl also contribute to an increasing yield.

Figure 7: Data from factorial design expriment plotted in a three dimensional matrix

Subsequently to the optimization process the resulting buffer condition 10 mM Tris, 1 % BSA, 90 mM NaCl, 5 mM MgCl2 containing EDTA free Protease inhibitor was used to test different dCas9 fusion constructs.

Test of a fusion-construct

After having optimized the buffer conditions for uniBAss dCas9 – fusion proteins were studied aiming on two points. First to gather information about the target site for the CRISPR/Cas system in a straight forward manner and secondly to study which of the high yield buffer conditions from displays the most robust readout if applied on various dCas9 – fusion proteins. The dCas9 - VP16 fusion contruct for gene activation was tested on different target on the human VEGF (Vascular endothelial growth factor) gene.

Figure 8: 3 different VEGF biotinylated oligos were used as targets for 3 dCas9-VP16 constructs containing the respective crRNA

Figure 8 shows the dCas9 - VP16 fusion proteins containing different crRNA targeting VEGF loci. This experiment aimed to examine whether there are different affinities between the different crRNAs. It can be seen, that the VEGF loci 1 and 3 behave similar, whereas the locus 2 reveals a higher ABTS readout in uniBAss. This indicates a higher binding affinity of the dCas9-VP16 construct to the VEGF locus 2 oligo.

Quantification of dCas9

To be able to quantify the amount of dCas9 binding to the oligos in the uniBAss ELISA a purified HA-tagged protein (FM) was used. As the ng/µl concentration of this protein is known, a dilution row was performed to get a standard to convert the ABTS readout in ng protein bound. Figure 9 depicts the ng of Cas9 or the different dCas9 constructs calculated via the formula(see in figure 9) of the standard dilution row.

Figure 9: Quantification of different Cas9 and dCas9 constructs

The uniBAss ELISA performed for figure 9 indicated that the fusion constructs are 3 - fold less expressed than the constructs with the Cas9 or dCas9 only. The comparison between a dCas9 completely lacking the nickase activity and a Cas9 having one active nickase shows no significant difference. However the yield of the dCas9 construct without any nicking activity is slightly higher.

Reason for truncating dCas9

With a size of 160 kDa, the dCas9 protein of the CRISPR/Cas system is the largest molecular tool among Zinc Fingers and Transcription Activator–Like Effectors. Difficulties that occurred during the light induced translocation of a dCas9 fusion construct in the nucleus indicated that the size of dCas9 might be a bottleneck for efficient genome engineering hence a truncation strategy was developed.

How to truncate dCas9

To reduce the size of dCas9 we ran a PCR over the backbone with two primers binding in dCas9, one with an overlap for gibson assembly. After the PCR one-fragment gibson cloning was performed. We tried five different strategies where to reduce the size of the dCas9 protein. In the first attempt we deleted 365 bp near the N-terminus of the protein. In a second try we erased 306 bp near the C-terminal part of dCas9. Here we assume the reverse transcription domain of the protein, which will probably be responsible for DNA binding. Therefore this truncation is thought to verify this assumption and can serve as a negative control. For truncation 3, 4, and 5 we just deleted the beginning, the middle and the end of dCas9 more drastically by throwing out about 1000 bp.

Results

The PCR over the pSB1C3 backbone of the dCas9 plasmid worked for all primer pairs. The different lengths of the PCR products, due to differently truncated dCas9, are clearly visible. After Gibson assembly with these PCR products the test digest of the plasmids also shows the expected length. We cut with NotI so that the insert of the plasmid is cut out. Only for truncation 4 we received a shorter fragment than expected. To ensure the expression of the shortened dCas9 version we transfected the midi-preps of them into HEK 293T cells and performed a western blot with the cell lysates. As it can be seen in figure 10 we could proof the expression of the standardized truncations. As expected truncations 1 and 2 are about 100 amino acids shorter resulting in an approximately 11 kDa shift in the western blot compared to the full-lengths dCas9. Truncation 3 and 5 run at around 120 kDa. Here about 300bp are missing, resulting in a size reduction of about 33kDa. Only truncation 4 is shorter than expected. It has about 80 kDa and the sequencing of the midi-prep of T4 revealed a nearly 2000 bp deletion in the dCas9.

Figure 10: Western blot with anti HA-antibody to show the expression of the different truncations.

After the proof of expression we tested the binding capacity of the truncated dCas9 versions to DNA with the uniBAss ELISA (see Results of uniBAss). By now we could not show binding of the untruncated dCas9 in pSB1C3 no matter of CMV or SV40 promoter. This is due to a very weak expression of dCas9 in pSB1C3 which results in amounts of dCas9 which are not sufficient for uniBAss detection levels. Because we cannot prove the binding of the full-length dCas9 in the iGEM backbone, we are not able to make a statement about the binding capacity of the truncated versions. At the moment we are trying to increase the amount of dCas9 for uniBAss to be above threshold.

References