Part:BBa_K2807000

rAPOBEC1

Usage and Biology

APOBEC, a cytidine deaminase enzyme, is a well-established protein which is known to perform C to U conversions on RNA strands (Smith, Bennett, Kizilyer, McDougall & Prohaska, 2012). Therefore, we intend to fuse an RNA-targeting Cas13 to the APOBEC enzyme. We hypothesised that the APOBEC enzyme, when fused with a Cas13 protein may perform a C to U conversion for a specific RNA target.

Figure 1. Diagram of the deamination of Cytidine to Uridine, which is accomplished by cytidine deaminases such as APOBEC.

Hence, our project aims to develop and verify an RNA editing system using the enzyme APOBEC fused to dCas13b. Deactivated Cas13b, which differs from the activated Cas13b by two amino acids (H133A and H1058A), is able to still be guided by guide RNAs (gRNAs) to target the specific RNA strand. However, as enzymatic activity is deactivated, no cleavage of RNA strands will occur. dCas13b together with the gRNA will guide the fusion protein to the appropriate site on the target mRNA to be edited. Then, the APOBEC will make the specific C to U edit on the mRNA strand.

Determining the activity of the rAPOBEC catalytic domain using in vitro DNA Deaminase Assay

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.The reactions were then incubated at 37°C overnight. The translated protein was then used for the DNA Deaminase Assay.

The DNA substrate used in this experiment were 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. The samples were then loaded onto a TBE-UREA PAGE gel for furhter analysis. The results of the SDS-PAGE gel electrophoresis is as shown below (Figure 2).

Figure 2. SDS-PAGE gel electrophoresis of the deaminase assay for rAPOBEC. There are faint bands at around the same size at all lanes except for lane 1 and 10 despite having both positive and negative controls. This would mean that the deaminase assay may not have worked to our expectations.

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 3). rAPOBEC protein has a size of approximately 25 kDa, however as shown in Figure 3, 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.

Figure 3. Western blot of rAPOBEC protein with T7 antibody. The expected band size of rAPOBEC is approximately 25 kDa. However the size of the band shown above is between 100 kDa and 130 kDa.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 103
  Illegal SapI site found at 625