Design And Construction Of A Far-red Light-controlled Transgene Expression System And Its Application Research

Optogenetics combines optics and genetics to control the activity,metabolism,and morphology of cells by light,as a method for precise spatiotemporal regulation,has been widely applied to multiple fields.However,at present,ultraviolet light and blue light play main roles in optogenetic tools,the vivo application of which is restrained due to the weak tissue penetration and poor biocompatibility.Although PhyB/PIF6 system regulated by red light and MrkH system regulated by far-red light in microorganism with better tissue penetration and low cytotoxicity have been developed,there are still some problems such as poor activation efficiency and extra need for co-factors.Therefore,this research aims to develop a new optogenetic system with strong tissue penetration,high induction efficiency,better compatibility and independence of exogenous co-factors,so that it can be applied to therapy for diabetes,optogenetic regulation of genome transcription,regenerative medicine and so on.First of all,we constructed and optimized STING-based Far-Red Light-triggered gene expression system?FRL-v1?by assembling FRL-activated c-di-GMP synthase-BphS,c-di-GMP sensor STING,putative FRL-dependent STING-responsive synthetic promoters(P_FRL1.x)and c-di-GMP–specific phosphodiesterase-YhjH,the inducible efficiency of which was up to 50-fold.Despite the fact that FRL-v1 system is conspicuously superior to the present optogenetic tools,its further application is limited due to the interference of other intracellular signals,since FRL-v1 system utilized the inherent STING pathway in mammalian cells.Hence,we reprogrammed the downstream of this system to develop a far-red light-mediated gene expression system with better anti-interference capability.BldD derived from Streptomyces coelicolor,which can form dimers in the presence of c-di-GMP and specificly recognize sequences containing bldM and whiG,was engineered to construct Far-Red light-inducible Transcriptional Activators?FRTAs?through fused with p65,VP64 and HSF1.And then Far-Red Light-triggered promoters(P_FRLx)was designed and constructed through combining whiG or bldM with minimal promoter P_hCMVminCMVmin or P_hCMVmin3G.Finally,by assembling the designed promoters and transcriptional activators with BphS and YhjH,we screen out a new Far-Red Light-mediated Transgene Expression System?FRL-v2?with much higher FRL-triggered induction ratios?61 fold?and better anti-interference.Better adjustability,reversibility,illuminated time and intensity-dependent manner and highly spatiotemporal specificity were achieved in the results of dynamic characteristics of FRL-v2 system both in vitro.Otherwise,FRL-v2 system,implanted into mice,presented strong tissue penetration,better compatibility as well as illuminated time and intensity-dependent manner.Then,we applied FRL-v2 system on diabetes therapy to overcome the deficiencies of traditional therapies such as short duration of pharmaceutical effect,cumbersome operations and so on.In this case,we developed light-controlled stable cell lines which are capable to secrete hypoglycemic drugs as response to FRL.By implanting these engineered cells into Type 1 and db/db diabetic model mice,the blood glucose level of diabetic mice was significantly decreased and stabilized at normal level for long time after illumination of FRL.In order to further improve the accuracy and convenience of diabetes treatment,we combined optogenetic cells with electronic engineering and software engineering to develop a SmartController Systemfor diabetes intelligent diagnosis and therapy semiautomaticly.The system was tested in db/db model mice to verify the possibility of blood glucose-dependent hypoglycemic drug release,that is,semiautomatic diagnosis and treatment of diabetes was achieved.To control genome transcription directly with FRL,we developed a Far-red light-Activated CRISPR-dCas9 Effector?FACE?device by combining FRL-v2 system with CRISPR-dCas9.Firstly,Synergistic Activation Mediator?SAM?system with better activation efficiency was selected to design and construct various MS2-based Far-red light-response Genome Transcriptional Activators?FGTAs?.After screening different FGTAs and P_FRLx,optimal FACE system was obtained.And then the function of FACE system was tested in vivo and in vitro,and genome transcription was successfully regulatedunder the illumination of FRL.To verify whether endogenous genes could be activated by FACE system,we introduced FACE system into induced Pluripotent Stem Cells?iPSCs?to activate single neural transcription factor NEUROG2 by far-red light.As a result,iPSCs were successfully differentiated into functional neurons.In conclusion,we designed and constructed a novel far-red light-mediated transgene expression system?FRL-v2?with strong tissue penetration,better compatibility,high induction efficiency and independence of exogenous co-factors to expand the optogenetic toolbox.At the same time,we first successfully combine electronic communication technology and optogenetic technology to apply on smart diagnosis and therapy for diabetes and offer innovative ideas for accuracy and individualization treatment of diabetes.Meanwhile,at the basis of FRL-v2 system we developed far-red light-controlled CRISPR-dCas9 system?FACE?,and made first successful attempt to directly regulate the expression of endogenous gene in mice by FRL,realizing the control of cell fate through FRL.We believe that FRL-v2 system and FACE system provide new tools with high temporal and spatial specificity for basic research,clinical research and translational medical research and new ideas and strategies for treating disease.And they also provide a step toward for personalized,precise,intelligent and global medicine in the future.