Stitching together complex protein fusion for epigenetic studies.
For this protein fusion project, our customer asked us to generate some tools to be used for precision epigenetic engineering. The core idea was to use Crispr/Cas9 system as a targeting system to address various DNA modifying enzymes to specific locations (i.e. promoter sequences of gene of interest), thus altering the DNA modification status of the targeted sequences. 20 different DNA modifying domains were considered.
It was also asked to include fluorescent protein as tracker so the transfected cells could be sorted to enrich the relevant population to analyze. In order to avoid interferences with the tested catalytic activities, the fluorescent marker had to be fused with an auto-cleavable peptide (T2A). At last, the fusion protein was also tagged with several peptide-tags, so it could be purified from cell lysate for in vitro activity analyses.
Eventually, the sum of all requirements resulted in the fusion of 10 coding sequences of various sizes to be fused, in frame, with high precision (Figure 1). We Assembled these vectors using our one step/one tube reaction technology applied to a mix of DNA bricks recapitulating all the desired sequences. Moreover, the Bricks were also designed so it was easy to substitute specific parts (catalytic domains or fluorescent proteins) to generate further variability at later stages.
Figure 1: from needs to tool. The Fitting vector was built in a complete customizable way once the requirements were defined through a co-design approach involving the research team and e-Zyvec’s experts. Designed with d-Zyvec.
Studied cells were co-transfected with the ‘Fusion’ vector and a sgRNA vector to assess the enzymatic activity. In the figure 2 below, 3 different sgRNA vectors (also assembled using our method) were compared. The single guide RNAs were chosen to cover different parts of a specific promoting sequence (figure 2A). Co-transfected cells were harvested to analyze the epigenetic status of the promoting sequence. Figure 2B shows that co-transfection of the fusion vector with the sgRNA-2 vector does indeed alter the epigenetic status of the targeted sequence.
Figure 2: Using the tools. Several co-transfections were realized on the studied cells to assess the efficiency of the system.
Experimental results: courtesy of Dr Audrey Vincent, Canther Laboratory, Lille France. Study funded by Cancéropôle Nord-Ouest.