Part:BBa_K5191000

Cytosine Base Editor-T7 RNA ploymerase

rAPOBEC1-RNA Polymerase fusion gene

rAPOBEC1-RNAP fusion protein is a biotechnological tool that combines a cytidine base editor (CBE) with RNA polymerase (RNAP). This fusion allows for the targeted editing of DNA sequences by enabling the conversion of specific cytidine residues to thymine in DNA. The rAPOBEC1 component utilizes a deaminase enzyme to facilitate this conversion, while the RNAP component is responsible for synthesizing the RNA. By integrating base editing capabilities with RNA synthesize, rAPOBEC1-N terminal RNAP-C split RNAP represents a powerful strategy for gene editing.

CBE or cytosine base editor is the foundational basis of our system. Its a mutagen that mutates specific DNA nucleoside. The role of CBE in the first generation mutagenesis system construct is targeted DNA editing. The T7 promoter initiates transcription in the presence of T7 RNA polymerase, focusing editing to specific regions. The LacO operator allows for inducible control via IPTG, while the RBS boosts translation of rAPOBEC1, a cytidine deaminase that converts cytidine (C) to thymine (T) in DNA. The N-terminal and C-split RNAP components enable RNA synthesis, and the 8xHis tag facilitates protein purification. Ampicillin resistance serves as a selection marker, ensuring identification of successful transformations

The construction of the second generation mutagenesis system utilizes a T7 promoter-LacO-RBS system to drive expression of the rAPOBEC1-nMagHigh1 and pMagHigh1-C-split RNAP fusion proteins, which are central to targeted gene editing. Similarly, rAPOBEC1 is a cytidine base editor (CBE) that converts cytidine (C) to thymine (T), enabling precise DNA editing. nMagHigh1 and pMagHigh1 interact under blue light, reconstituting the split RNAP, which drives transcription. This system provides tight temporal control over the editing process, allowing C-to-T mutations only during specific time windows. The addition of LacO for IPTG induction and blue light regulation enhances the control of gene editing, making the second-generation system more precise and efficient for controllable mutagenesis.

Figure 1: A) The statistical analysis of the difference in base pair mutations between the first and second generation

To compare the mutation frequencies induced by the second-generation black box (CBE-Mag-RNAP) and the first-generation black box (CBE-RNAP), we separately transformed both plasmids into E. coli BL21, using continuously expressed mCherry as an internal control (Figure 1A). We established a control group (where water was added to prevent mutation activation), a second-generation black box group (where IPTG was added and placed under blue light to activate mutations), and a first-generation black box group (where IPTG was added to trigger mutations). We then used fluorescence-activated cell sorting (FACS) to analyze the three groups based on GFP and mCherry fluorescence intensities, isolating cell populations with increased and decreased fluorescence levels (Figure 1B). The results (Figure 1C) showed that the percentage of cells with increased fluorescence in the second-generation black box group (14.78%) was significantly higher than in the first-generation group (3.42%) and the control group (11.95%). This demonstrates that the second-generation black box greatly enhanced mutation frequency compared to the first generation, with most mutations being C-to-T substitutions (Figure 1D). This experiment proves that oursecond-generation of black box which was created based on the first generation effectively increasing the mutation frequency.

Figure 1: A)Plasmids of the second-generation black box and the first-generation black box. B) Fluorescence-activated cell sorting (FACS) based on the fluorescence intensities of GFP and mCherry. C) Proportion of cells with increased and decreased fluorescence intensity in the three groups. D) Statistical analysis of the proportions of different types of random mutations.

Usage and Biology

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]