最先端研究セミナー

演題：　Development of genome editing and gene regulation systems utilizing specific DNA binding domains

Abstract:

Genome editing platforms, such as ZFN, TALEN, and CRISPR-Cas9, are powerful tools for gene manipulation. Given that these platforms possess a wide variety of promising applications in medical science and biotechnology. Inducible association of protein domains by stimuli such as light or chemicals has widely been used for the temporal control of enzymatic activity. As the first topic, a chemically inducible nuclease system utilizing the association of FKBP-FRB by rapamycin was designed and constructed. The DNA-binding and FokI domains of ZFN or TALEN were split at the linker region, and then fused with FKBP and FRB, respectively. The split-type nuclease was used as a pair with intact nuclease at the target site. Some combinations of split-type nucleases showed induced cleavage by rapamycin addition. It was also shown that the concept of split-type nuclease can also be applied to nuclease-dead Cas9 (dCas9)-FokI system. The second topic is the use of DNA binding specificity of genome editing tools for gene regulation systems by combining these tools with transcription-activator or -repression domains. Targeting the gene promoter sequence using multiple artificial transcription factors synergistically and efficiently regulate gene expression. In this study, the rapamycin-inducible system was applied for gene regulation by using multiple TALEs and dCas9. We constructed FKBP-tagged TALEs and dCas9; the effector domains with FRB, VP64 for activation and SID×4 for repression, were also prepared. The fold change in gene expression was evaluated by luciferase reporter assays. The results showed that rapamycin induced FKBP-FRB association and that gene expression was regulated according to the presence of the FRB-effector fusions. The system was further applied for controlling endogenous gene expression in 293A cells. The results revealed that the expression could be regulated by rapamycin in a time-dependent manner. Finally, GAI-GID domain association was used for orthogonal gene regulation. The system could enable the control of gene function on cellular phenomena owing to the programmable timing of expression and differential expression of multiple genes.

As the third topic, we are developing a recombinase system for genome editing, especially for large gene deletion and gene integration [1]. Current system utilizes zinc finger domains as a specific DNA binding domain. Advantageous points of the recombinase system over currently available nuclease-based system will be introduced.

Reference

[1] Nomura, W., et al. (2012) Biochemistry 51, 1510-1517.

担当分野：　生体機能分子合成学分野 大塚（内線：4620）

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