Enhanced Gene Disruption at Specific Promoter Region By Simultaneous Digestion of ZFN or Crispr/Cas System

Artificial zinc finger proteins (ZFPs) consist of Cys₂-His₂-type modules composed of about 30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes, such as nucleases, recombinases [1], and methylases [2], with programmable sequence specificity. Genome editing and modification by the enzymes could be applied for many fields of basic research and medicine. Recent findings of new platforms for genome editing such as transcriptional activator-like effector (TALE) and CRISPR/Cas system expands the possibility of the technologies. These technologies enable efficient knock-out of gene of interest or regulation of gene function. In this study, ZFN pairs targeting promoter region of human telomerase reverse transcriptase (hTERT) were designed and constructed. Endogenous gene targeting by the paired ZFN showed successful induction of mutation at the upstream- and downstream-site of core promoter region of hTERT. The simultaneous digestion by both pairs of ZFNs showed enhanced disruption of the target gene. Guide RNAs of CRISPR/Cas system targeting the same sequence with ZFN were also constructed and tested. The enhancement of gene disruption by the simultaneous digestion at the upstream- and downstream-sites was observed as shown in the ZFN experiments. Cells with gene disruption at the core promoter region of hTERT showed decreased expression of hTERT. The study will provide the information for the role of hTERT promoter activity related to cell-specific phenotypes such as immortality of cancer cells. Furthermore, the gene targeting for specific promoter region could be applied for studies in Synthetic Biology.

[1] Nomura, W., Masuda, A., Ohba, K., Urabe, A., Ito, N., Ryo, A., Yamamoto, N., Tamamura, H. Biochemistry, (2012) 51, 1510-1517.
[2] Nomura, W., Barbas, C. F., III J. Am. Chem. Soc., (2007) 129, 8676-8677.