Inspired by the work of Martin et al. (2018), as well as WPI Worcester 2016 iGEM project, we designed an EGFP-T2A-mCherry dual fluorescence reporter plasmid system. This plasmid consists of two fluorescence markers, an enhanced green fluorescent protein (EGFP) gene and a mCherry gene (Figure 1). Here, mCherry is expressed constitutively and used as a marker for transfection efficiency and the overall fusion protein expression level. The EGFP gene is modified such that the start codon of EGFP is mutated (ATG → ACG), resulting in a truncated GFP that is non-functional (Figure 1). Since the minimal fluorescence region for EGFP is from amino acid 7-229 (Li et al., 1997), this truncation mutation will cause the production of non-functional EGFP protein, while mCherry expression is not affected. However, when this mutation (ACG → ATG) is corrected by our RESCUE Editor system, the normal expression of EGFP is restored and can be visualised either on a fluorescence microscope or via flow cytometry.

Both the mCherry and the EGFP genes in the fusion protein are linked by a self-cleavage peptide, T2A. When the ribosome reaches the T2A oligopeptide during translation, it will fall off from the mRNA and translation would be restarted for the next fluorescence gene. We chose T2A among the 2A family peptides as it has the highest cleavage efficiency (Liu et al., 2017). This is to ensure that expression of EGFP and mCherry are under the control of the same promoter, such that the level of mCherry fluorescence is linearly correlated to the level of EGFP fluorescence. In this way, the mCherry expression would be a useful indicator to normalise the expression of the dual reporter system between individual cells in a transient transfection experiment.

The relative number of editing events can be quantified by the percentage of dual-colored cells transfected with mutant ACG reporter as compared to cells transfected with the WT reporter, or relative green fluorescence intensity after normalizing to the red fluorescence intensity. We will be testing our reporter system in both E. coli and transfected HEK293T cells.

A further modification to the original EGFP and mCherry sequence is a six amino acid truncation at the N-terminus of EGFP and a seven amino acid truncation at the C-terminus of mCherry. Since EGFP and mCherry have the same nucleotide sequence for the first 22 and last 23 nucleotides, such truncation makes cloning easier. Moreover, since bacteria like E. coli can utilize alternative start codon GUG up to 13% of the time (Hecht et al., 2017), and the first 18 nucleotides of EGFP sequence contain one GUG, this truncation mutation will eliminate the chance of background translation initiation in bacteria, thus making the reporter suitable for both bacterial and mammalian systems.

The second reporter design involves a GFP fusion protein linked to the human GLB1 gene. GLB1 encodes a human lysosomal acid beta-galactosidase, an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. Genetic mutations in the GLB1 gene have been seen in the occurrence of autosomal recessive lysosomal storage diseases such as GM1 gangliosidosis and Morquio B disease (Suzuki et al., 2001). There are more than 130 single base mutations that have been identified in GLB1 and some of these mutations are the (T → C) base change as listed in Table 1. Hence the use of our RESCUE Editor system could potentially restore the function of GLB1 in patients with this mutation, thus acting as potential therapy towards such genetic diseases.

As such, we designed an EGFP-GLB1 fusion protein to act as an alternative base editing reporter for potential testing in a disease model in the future. Based on published work on GLB1 mutation genotyping in patients and its corresponding residual enzymatic activity (Table 1), we carried out site-directed mutagenesis on 319T → C, which leads to phenylalanine at amino acid 107 to be changed to leucine (TCT mutant), and on 1166T → C, which leads to leucine at amino acid 389 to be changed into proline (CCG mutant). These two single nucleotide substitutions are expected to result in total loss or minimal residual beta-galactosidase enzymatic activity.

Using colourimetric (X-gal, ONPG) or fluorescent (DDAOG) beta-galactosidase substrate, we can then check for the restoration of GLB1 enzymatic activity after base editing in transfected cells. A successful base repair by our RESCUE Editor system would allow the conversion of a mutant GLB1 to a functional GLB1, thus leading to restoration of enzymatic function (Figure 2). At the same time, the EGFP reporter can act as transfection/expression control for internal normalisation.