Team:Freiburg/Project/method

uniBAss - uniCAS Binding Assay

How uniBAss works

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 the HEK293 cells are taken up in diluent buffer and are lysed by sonifying. Then the 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 washing away everything that did not bind to the oligos 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 in color.

6. The intensity of the color is than measured at 405nm and represents the amount of dCas9 bound in each well.

Results

Fig. x: uniBAss results showing dCas9 amounts binding to target oligos for each construct.

Development of uniBAss

uniBAss Introduction

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 Immuoprecipitation (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, the 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 this 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 Cas9 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.

Fig. x: Binding restrain of the Cas9 MX1 crRNA complex for varying MgCl2 conditions

The figures show that the binding behaviour of the dCas9 / crRNA EMX1 strongly depends on the salt concentrations.

Fig. x: Binding restrain of the Cas9 MX1 crRNA complex for varying NaCl conditions

This experiment 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.

Fig. x: Design of the different oligos

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 consiting of the crRNA locus is essential to get the dCas9 to bind. The presence of a second long sequence at the 3’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 studied was focused 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.

Fractional design

The figure shows that the binding behaviour of the Cas9 / 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.

Fig. x: Factorial design expriment

The plotting of the data in 2 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 3 dimension (figure xxx) it occurred that the decreasing amounts of NaCl also contribute to an increasing yield.

Fig. x: Factorial design expriment

Reason for truncating dCas9

With 160 kDa is the dCas9 protein of the CRISPR/Cas system 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 performed. We tried five different strategies where to reduce the size of the dCas9 protein. In the first attempt we deleted 365bp near the N-terminus of the protein. In a second try we erased 306bp 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 1000bp.

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 HEK293t cells and did western blot with the cell lysates. As it can be seen in imagexx 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 25kDa shift in the western blot. Truncation 3 and 5 run at around 120kDa. Only truncation 4 is shorter than expected. It has about 80kDa and the sequencing of the midi-prep of T4 revealed a nearly 2000bp deletion in dCas9.

Fig. x: 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 bind 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 version. At the moment we are trying to increase the amount of dCas9 for uniBAss to be above threshold.

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