Design And Construction Of A Light Inducible Endogenous Gene Transcriptional Activation System For Cell Reprogramming Into Pluripotency

Stem cells have properties of self-renewal and differentiation to multiple cell lineages,which hold great promise for cell differentiation mechanisms,disease model construction and regenerative medicine research.Embryonic stem cells(ESCs)can only extract from embryos,which limited the clinical application.Induced pluripotent stem cells(i PSCs)overcome the limitation in use of ESCs and problems of the ethical issues and immunological rejection with ESC,which has greatly promoted research in regenerative therapy.At present,mouse embryonic fibroblasts or human foreskin fibroblasts are reprogramed into i PSCs mainly through exogenously constitutive expression of transcription factors such as Sox2,Oct4,c-Myc and Klf4.However,there is a risk of tumorigenicity and uncontrollability for reprograming somatic cells into i PSCs by over-expressing exogenous genes in a virus-mediated manner.Using antibiotics as inductors to regulate the activation of endogenous gene expression for i PSCs reprogramming also limited by drug regulation system.It is still an important issue that how to precisely regulate the formation of i PSCs by activating endogenous transcriptional factors.Utilizing the concept of synthetic biology,combining the precise targeting endogenous genes with CRISPR/d Cas9 gene editing technology and the spatiotemporal specificity,reversibility and non-invasive advantages of optogenetics technology,we designed and constructed a blue light inducible CRISRP/d Cas9 system for endogenous gene transcriptional activation(BICE),Which is expected to solve the problems of tumorigenicity,uncontrollability and low efficiency during i PSCs formation.The BICE system consists of three parts: light-controlled binding elements,transcriptional activation complex,and d Cas9-sg RNA complex.The light-controlled binding element is composed of Tet R-CIBN and CRY2-VP64.CRY2 and CIBN are cryptochromes and cryptochromes interacting b HLH1 proteins,both of which are expressed by the EF1?promoter.Under blue light irradiation,CIBN and CRY2 bind to each other,and the complex(Tet R-CIBN-CRY2-VP64)enters the nucleus and approaches Ph CMVmin*-1promoter through the interaction of Tet R and Tet O7.VP64 acts as a transcription activator activating the transcription of complex MCP-VPR(MCP-VP64-p65-RTA)which is downstream of the Ph CMVmin*-1.d Cas9 and g RNA are initially expressed by the EF1? promoter and U6 promoter,respectively,and bound to the targeting sequence.The MCP-VPR produced by blue light-controlled transcriptional activation targets the downstream of genes through the interaction of MCP and MS2 which included in g RNA sequence,initiates the expression of endogenous genes Sox2 and Oct4.In order to optimize BICE system,we first designed and constructed multiple g RNA combinations targeting Sox2 and Oct4 and three different transcriptional activation complexes.We found that the BICE system with the concatenated combination of sg RNA and MCP-VPR transcription complex show robust activation of endogenous targeted genes.And then,gene expression kinetics and reversibility of BICE system are also studies.During BICE system mediated MEF cells reprogramming into i PSCs,Sox2 and Oct4 was significantly upregulated at 4 days under blue light illumination,and the pluripotency genes Nano G,Esrrb,Fgf4 and Nr5a2 also increased with time,at levels similar,but not identical,to those of i PSCs cell line at day 21.Immunofluorescence staining show that the pluripotency markers Sox2,Oct4,SEEA-1,TRA-1-60 were significantly expressed in the i PSCs formed under blue light illumination,while none of these markers was expressed in the dark group.In addition,light-controlled i PSCs were morphologically similar to mouse ES cells and stained positive for alkaline phosphatase.Transcriptomic analysis also showed the i PSCs reprogrammed from MEF under blue light illumination have similar transcriptome expression profiles with i PSCs cell lines.Finally,in order to verify that the light-controlled i PSCs differentiate into all cell types we injected the i PSCs subcutaneously into NOD/SCID mice.The light-controlled i PSCs formed teratomas and histological examination of the teratomas revealed three germ layers,which suggests that the i PSCs formed by BICE system have the ability to differentiate in vivo.In summary,the BICE system can successfully reprogram MEF cells into i PSCs.This light-controlled method solves the shortcomings of serious drug diffusion in vivo,irreversible induction,and harmful to the body in drug-induced reprogramming,provides a new idea and new strategy for the formation of i PSCs,which used in regenerative medicine research and clinical treatment.