The rAPOBEC deaminase assay was conducted to determine whether the catalytic domain of the rAPOBEC is functional. The rAPOBEC used in our construction of the fusion protein only contains the catalytic domain of the enzyme. This domain would catalyse the conversion of Cytosine to Uracil in RNA strands as shown below (Komor et al., 2016).
The in vitro DNA deaminase assay was conducted to determine whether the rAPOBEC catalytic domain of the RESCUE editor is functional.
In vitro transcription and translation of rAPOBEC was carried out using Promega’s TNT® Quick Coupled Transcription/Translation Systems. 1 μg of plasmid (rAPOBEC in pGEM-T Easy vector) was added to 40 μL TNT® T7 Master Mix and 1 μL of 1 mM Methionine. Nuclease-free water was topped up to a final volume of 50 μL for each labelled tube. The reactions were then incubated at 37°C overnight. The translated protein was then used for the DNA Deaminase Assay.
A deaminase assay master mix was prepared for 11 reactions as shown from the table below (Table 1). 9 μL of the master mix was pipetted into each tube, followed by 5 μL of rAPOBEC protein. The DNA substrate used in this experiment was ordered from Integrated DNA Technologies and labelled with the Cy3 dye with the following sequence /5Cy3/TTGAGACATACGCGATACAATTTGATCAGTATATTAGAGA. A negative control was also prepared where no protein was added to the tube. The reactions were incubated at 37°C for two hours.
Team:NUS Singapore-Sci/Editor Results
Editing System
Results
1. Determining the activity of the rAPOBEC catalytic domain using in vitro DNA Deaminase Assay
| Deaminase assay components | Amount per reaction (μL) |
|---|---|
| Nuclease-free water | 3.5 |
| 10x deaminase buffer | 1 |
| Cy3 labelled DNA substrate (10 pmol/μL) | 0.5 |
Next, Uracil DNA glycosylase (UDG) master mix was prepared as shown in Table 2. 5 μL of the UDG master mix was added to 10 μL of each of the deaminase assay reaction tubes. The reactions were incubated at 37°C for 45 minutes.
| UDG Enzyme Mix | Amount per reaction (μL) |
|---|---|
| Nuclease-free water | 2.75 |
| 10x UDG buffer | 1.5 |
| USER enzyme (1 U/μL) | 0.75 |
After 45 minutes, 5 μL of 0.6M NaOH was added to each tube and incubated further at 37°C for 20 minutes. After incubation, 5 μL of 6X DNA loading dye was added to each tube and heated at 95°C for 3 minutes and immediately placed on ice. 15 μL of each sample was then loaded onto a TBE-UREA PAGE gel and resolved by electrophoresis at 200V for 30 minutes at room temperature (25°C) in 1x TBE buffer. The results of the SDS-PAGE gel electrophoresis is as shown below (Figure 1).
The rAPOBEC protein is expected to carry out deaminase activity to convert Cytosine to Uracil on the Cy3 labelled DNA substrate. The addition of the USER enzyme would result in the cleavage of DNA substrate resulting. As such, we expect to see two bands on the gel, one of a bigger size and one of a smaller size. Since the deaminase assay results only showed one single band across all the lanes, we used Western blot to check for the size of the protein using T7 antibodies (Figure 2) after the rAPOBEC part was cloned into pGEMT Easy vector under T7 promoter. rAPOBEC protein has a size of approximately 25 kDa, however as shown in Figure 2, there seems to be a band between 100 kDa and 130 kDa. This could mean that rAPOBEC could have formed protein aggregates or the protein that was translated was not rAPOBEC.
2. Expression of Cas13b and dCas13b in mammalian cells
To verify the expression of the Cas13b part in the mammalian system, HEK293T cells were transfected with px330A-PspCas13b and px330A-dPspCas13b using Lipofectamine 2000. Cas13b is able to target and cleave messenger RNA strands with the aid of a compatible guide RNA, while the nuclease activity in dCas13b has been inactivated through the mutation of the enzymatic catalytic sites. An inactivated form of Cas13b is important for our RESCUE editor as we aim to make a site-directed change rather than cleave the target strand.
Transfected cells were lysed and the cell extracts were separated on an SDS-PAGE gel. As our PspCas13b and dPspCas13b includes an hemagglutinin (HA) epitope tag at the C-terminus, we were able to detect for either Cas13b and dCas13b protein using a monoclonal HA antibody through Western blotting. As both Cas13b and dCas13b are 3402 base pairs long, we expect a protein size of approximately 124 kDa to be expressed.
Transfected cells were lysed and the cell extracts were separated on an SDS-PAGE gel. As our PspCas13b and dPspCas13b includes an hemagglutinin (HA) epitope tag at the C-terminus, we were able to detect for either Cas13b and dCas13b protein using a monoclonal HA antibody through Western blotting. As both Cas13b and dCas13b are 3402 base pairs long, we expect a protein size of approximately 124 kDa to be expressed.
From Figure 3, the appearance of a band between 150 kDa and 100 kDa showed that both Cas13b and dCas13b were expressed at the right molecular weight. However, we note that the different intensities in protein expression across the three replicate samples may be attributed to variation in transfection efficiency across each sample.
3. Supplementary Information
| 10x deaminase buffer (1 mL) | Volume (μL) |
|---|---|
| 1 M Tris-HCl (pH 8.0) | 100 |
| 5 M NaCl | 100 |
| 1 M Dithiothreitol (DTT) | 10 |
| Water | 790 |
Recipe for 10X UDG reaction buffer:
200mM Tris-HCl (pH 8.2 at 25°C)
10 mM Ethylenediaminetetraacetic acid (EDTA)
100 mM NaCl
Supplied with Uracil DNA glycosylase
200mM Tris-HCl (pH 8.2 at 25°C)
10 mM Ethylenediaminetetraacetic acid (EDTA)
100 mM NaCl
Supplied with Uracil DNA glycosylase
References
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420-424. doi:10.1038/nature17946