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Team:Freiburg/Project/method
From 2013.igem.org
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<p id="h3"> | <p id="h3"> | ||
Reason to truncate Cas9 | Reason to truncate Cas9 | ||
| + | </p> | ||
| + | <p>With 160 kDa is the Cas9 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 Cas9 fusion construct in the nucleus indicated that the size of Cas9 might be a bottleneck for efficient genome engineering hence a truncation strategy was developed. | ||
</p> | </p> | ||
<p id="h3"> | <p id="h3"> | ||
How to truncate Cas9 | How to truncate Cas9 | ||
| + | <p> To reduce the size of cas9 we run PCR over the backbone with two primers binding in cas9, one with an overlap for gibson assembly. After the PCR one-fragment gibson cloning performed. we tried five different strategies where to reduse the size of the cas9 protein. In the first attempt we deleted 365bp near the N-terminus of the protein. In an second try we erased 306bp near the C-terminatl part of cas9. 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 cas9 more drastically by throwing out about 1000bp. | ||
| + | </p> | ||
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2013/a/a8/Freiburg2013_Plasmid_Cas9truncated.png" height="60px"> | <img src="https://static.igem.org/mediawiki/2013/a/a8/Freiburg2013_Plasmid_Cas9truncated.png" height="60px"> | ||
Revision as of 20:07, 29 September 2013
uniBAss - uniCAS Binding Assay
uniBAss ELISA
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 Cas9 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 Cas9 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 Cas9.
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 Cas9 bound in each well.
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 Cas9 - VP16 or Cas9 – KRAB constructs, but it remained unclear if the binding characteristics of Cas9 - VP16 / KRAB are different from Cas9 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 Cas9 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 Cas9 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.
Results
Cas9 truncation
Reason to truncate Cas9
With 160 kDa is the Cas9 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 Cas9 fusion construct in the nucleus indicated that the size of Cas9 might be a bottleneck for efficient genome engineering hence a truncation strategy was developed.
How to truncate Cas9
To reduce the size of cas9 we run PCR over the backbone with two primers binding in cas9, one with an overlap for gibson assembly. After the PCR one-fragment gibson cloning performed. we tried five different strategies where to reduse the size of the cas9 protein. In the first attempt we deleted 365bp near the N-terminus of the protein. In an second try we erased 306bp near the C-terminatl part of cas9. 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 cas9 more drastically by throwing out about 1000bp.
Results
Sources
