Team:BGU Israel/Part improvement
1. Our part is shorter (378 bp vs 459 bp), thus permitting more flexible design in various scenarios when construct length could be a critical issue.
2. Our part adds extensive characterization results in human cancer cells, showing data of promoter validation, and, importantly, functional data where hTERT promoter was used to successfully control the expression of dCas9-VP64 in cancer-specific CRIPSR-based activation system. In the original part, actual experimental data is missing.
Specifically, we show:
1) GFP expression under short human TERT promoter exclusively in cancer cells (Figure 1).
Fig.1. GFP expression under short human TERT promoter in human cancer cells (fibrosarcoma) and healthy fibroblasts, after calcium phosphate plasmid transfection (A) or AAV transduction (B). Bar: 200 micron.
2) GFP expression under synthetic activation promoter after the successful activation of Boomerang system (consisting dCas9-VP64 - under the control of hTERT promoter, guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter, and GFP under synthetic activation promoter) (Figure 2).
Figure 2. GFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter. Bar:100 micron.
3. The engineered design of hTERT promoter in cancer-specific CRISPR-based gene knock-out is also shown, further expanding the usability of the part (Figure 3).
Figure 3. A. Plasmid map of AAV vector expressing SaCas9 under the control of human short TERT promoter. B. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.