Design of Experiment

We have previously demonstrated utility of our tCas9 in the context of VPR fusion for gene activation. Next, we seek to expand on the potential utility of our tCas9 with other fusion proteins. The APOBEC1 fusion as described by Komor et al. (2016) in 3rd generation BE3 stands at close to 700bp, fused to the N-terminus of an nCas9. A 250bp uracil-DNA glycosylase inhibitor (UGI) is also fused to the C-terminus to improve editing frequence. The resulting BE3 construct gene is over 5kB, greatly exceeding the packing limit of rAAV viral vector ideal for in-vivo delivery. Thus, there is value in getting tCas9 variants to work with the BE3 variants.

We tested a model tCas9 variant, ∆Rec3, with the BE3 fusion. The ∆Rec3 fusion was selected for this preliminary experiment for two reasons. First, the ∆Rec3 truncation has been shown to still maintain considerable DNA binding affinity, with over 50% of WT dCas-VPR gene activation in reporter assays (NTU Singapore iGEM, 2017). Second, the Rec3 domain has been shown to play a key proofreading step to allow for HNH cleavage. This is of concern as the high efficiency of both base editors are dependent on HNH nicking of the non-edited DNA strand, promoting repair to match the edited strand. In an experiment, both the tCas9-BE3 fusion protein and its guide RNA are expressed in HEK293t cells, and the target site will be amplified and seuqenced to determine the percentage of modification.

Results and Analysis

Results indicate that there is a big drop in base editing efficiency when Rec3 has been truncated (Figure 1). In addition to the poorer DNA binding affinity as observed in gene activation experiments, the loss of Rec3 is likely to have led to loss of the necessary proofreading step required for HNH nicking, leading to only second generation BE2 functionality for efficiency of editing. 

Figure 1. ∆Rec3-BE3 compared against background (Empty) and WT-BE3. 

Conclusion

Results indicated the importance of maintaining HNH nickase activity for optimal base editing efficiency. The removal of Rec3 led to loss of HNH nuclease activity in the context of base editors. In addition, Rec3 participates in interaction with target DNA:gRNA heteroduplex, and the loss of which probably further decreased binding affinity of tCas9. This could explain the drop in base editing efficiency. Moving forward, other truncation sites could be tested to confirm the necessity of function Rec3 and HNH domains. 

Results also suggests that base editing is stricter than gene activation in requiring high affinity to the target DNA. This is not unexpected as base editing would likely require longer dwell time for the deamination reaction to be catalysed. The improvements achieved with our ∆3ple in this year’s project could potentially solve this issue, although HNH may have to be replaced to maintain nickase function. 

 Experimental Design

The adenine base editor (ABE) has been evolved from a transfer RNA adenosine deaminase (TadA) that capable of converting A•T to G•C base pairs to treat diseases caused by single-base mutations in the genome such as sickle-cell anemia. The ABE can then be fused to a dCas9 to target a desired site to edit the DNA guiding by sgRNA.

However, overexpression of a protein can lead to toxicity issue, especially with the large size of dCas9. In order to have a tight control of gene regulation, we construct two plasmids where TadA*-dCas9 is expressed by arabinose under pBAD promoter and sgRNA is expressed by IPTG under lac operator. The TadA*-dCas9 plasmid consists of chloramphenicol resistance while the sgRNA plasmid consists of a gentamycin resistance and a kanamycin resistance with a premature stop codon.

Figure 2. Two constructs to be transformed into E. coli

In order for the bacteria to survive on Chloramphenicol + Gentamycin + Kanamycin condition, the TadA*-dCas9 need to restore the premature stop codon of Kanamycin.

We transformed bacteria BW25141 with these two plasmids and used 2 hours of LB recovery under four conditions: no induction, 100mM arabinose, 500μM IPTG, and 100mM arabinose + 500μM IPTG. The samples were then spread on a Chloramphenicol + Gentamycin plate for positive control and a Chloramphenicol + Gentamycin + Kanamycin plate for selection.

Results and Analysis

The result of the transformation are shown in figure below. Colonies grown on chloramphenicol+gentamycin plate show that the host cells contained TadA*-dCas9 and sgRNA plasmids, but colonies only grown on chloramphenicol +  gentamycin + kanamycin plate prove the tightness of regulation of both promoters.

Figure 3. Survival of colonies after transformation and induction

As seen from above, similar growth was observed on control plates without the selection pressure of Kanamycin, suggesting a similar transformation efficiency. However, in the presence of Kanamycin, only the experiement with both inducers added showed siginificant growth, suggesting that both constructs are necessary for bacteria to grow in Kanamycin and proving the editing activities of the TadA*-dCas9 construct. Such a growth can be more rigorously quantified as survival frequency, defined as the ratio of the number of colonies on the Chloramphenicol + Gentamycin + Kanamycin plate to that on the Chloramphenicol + Gentamycin plate. We also collaborated with NUSGEM this year to validate our TadA*-dCas9 construct.