The Development Of Novel Synnotch System And Combining With CRISPR/Cas9 For Genes Regulation

The aim of synthetic biology is to make biology more efficient,reliable and at the same time able to adapt more treatments and researches.Thus,how to design a flexible and editable program for creating new genetic circuit systems is a vital problem of synthetic biology.The synNotch system which develops from the Notch signal pathway is a new sensing receptor system.It has the characteristics of regulatory,simplicity,universality and powerful editability,which are the powerful tools for cell engineering.By employing synNotch system in T cell engineering,it can increase the precision,effectiveness and safety of customized cell therapy while ameliorating the "cytokine storm" side effects and improving the therapeutic effects in solid tumors of traditional CAR-T system.In addition,the synNotch system also can be applied in the field of developmental biology,and other studies based on cell-cell contact.The synNotch system is a powerful platform,thus exploiting synNotch system and expanding its applications will be very significant.In previous studies about the synNotch system,we noticed that there wasn't enough expression of downstream target genes when the original version of synNotch(Mouse Notch1 core)was activated,which will sometimes limit its applications.Considering the issues of the exclusion and crosstalk between different species,would there be any different versions of synNotch to choose?The Notch signaling pathway shows high cross-species conservation in both invertebrates and vertebrates.Thus,we hypothesize that the Notch receptors in different species or the different subgroups in the same species might have different performance in the synNotch system.Therefore,based on the synNotch system created by Lim's team,we developed more versions of synNotch systems with different efficacy compared with the original system.Then,we optimized them by adding Gal4,fusing PEST and extending EGF.These new versions of the synNotch system might serve as an extension and enrichment for the original synNotch system,filling some deficiencies and limitations,and providing more options for the application of the synNotch system.At the same time,we also performed experiments on the regulatability,flexibility,editability and specificity on both new and original versions.The data demonstrated the superiority and flexibility of these synNotch systems.In addition,currently in the field of synthetic biological therapy,how to transfer the therapeutic signal from the outside to the inside of the cell and achieving the regulation of endogenous genes is still an issue that remains unaddressed.After proving the flexibility,orthogonality,and the strong editability of the synNotch system,we combined synNotch system with Cas9/Cas9:p300 system to develop a sensing platform which depends on cell-cell contact for endogenous genes regulation(knockout and activation),which is potential to solve the problem of achieving the conversion of therapeutic signals through the intercellular sensing pathway in synthetic biology.In other words,the engineering cells transport the output gene modules basing on the principles of synthetic biology can activate genes regulation(knocking out the key genes or activating therapeutic factors,etc.)when targeting tumor cells or reaching disease tissues,achieving the purpose of precision therapy or functional repair.This new system and designing concept are of great significance to the clinical application of synthetic biology.