Establishment Of An Adenine Base Editor And Pluripotent Stem Cell Lines With Low Safety Risks

Base editor-based gene therapy and pluripotent stem cell(PSC)-based regenerative medicine therapy are expected to become new approaches for disease treatment.But base editors have off-target risks,and stem cells have tumorigenic risks,both of which may lead to safety concerns in practical applications.In this study,we attempted to employ the strategies by introducing dual guiders into adenine base editors and a conditional expression suicide system into embryonic stem cells to solve the corresponding gene off-target problems and tumorigenic problems,respectively,thereby improving the safety of the two treatments and promoting their clinical application.The adenine base editor(ABE),consisting of adenine deaminase,nCas9,and gRNA enables efficient A-to-G base conversion at DNA target sites.However,ABE has obvious off-target editing effects,mainly including Cas9-independent RNA off-target effects and Cas9-dependent DNA off-target effects.However,only the issue of Cas9-dependent off-targets has not been effectively solved,so the safety problem of ABE clinical application still exists.To solve this problem,in this study,we devised a strategy to introduce dual guiders in the ABE,in which a transcription activator-like effector(TALE)was fused with adenosine deaminase,while the gRNA was fused with nCas9 to develop a novel base editing system named TaC9-ABE.In this system,nCas9 is guided to the target site by sgRNA,while the adenine deaminase is guided by transcription activator-like effector(TALE).Only when nCas9 is guided to the target site close to the TALE target to open the DNA duplex and nick the targeted strand,TALE-guided adenine deaminase can convert A to G on the on-target single strand.In the experiment,we established an efficient base editor names TaC9-ABE after multiple rounds of structural optimization(including the fusion site of deaminase and TALE,the length of the linkers,and the distance between TALE and gRNA,etc.).The results demonstrated that the TaC9-ABE system can efficiently edit multiple gene loci in different human cell lines.The optimal distance between the target sites of the dual navigators is within 6-12 bp,and the size of the editing window can be controlled by changing the distance of the recognition interval.The three gene loci of rabbit embryos OTC-1,OTC-2 and DMD were edited in vivo by prokaryotic injection,and the editing effect was close to or higher than that of ABE7.10.Besides,we performed deep sequencing of 40 Cas9-dependent off-target sites and 25 TALE-dependent off-target sites in 7 gene loci.And the results of the comparative analysis found that TaC9-ABE did not produce either Cas9-dependent or TALE-dependent off-target sites.But the classic ABE7.10 editor showed off-target editing at about 50%of predicted off-target sites.The results above suggest that the TaC9-ABE system can eliminate the Cas9-dependent off-target effects.Pluripotent stem cells have infinite proliferation ability and can differentiate into almost all types of cells,which have broad application prospects in the field of regenerative medicine.However,PSC-derived cells carry a risk of tumorigenicity after transplantation.To address this problem,in this study,we designed a novel strategy for OCT4-driven expression of inducible suicide gene caspase-9(OCT4-iCasp9).Since OCT4 is only expressed in pluripotent stem cells,and iCasp9 can initiate apoptosis under the regulation of chemical induction drug(CID),resulting in cell suicide.Therefore,under this strategy,CID can effectively remove residual pluripotent cells in tissues after transplantation without affecting the survival of differentiated functional cells,thus establishing a new safety strategy for stem cell therapy.In our experiments,we established four human and mouse pluripotent stem cell lines(H9P,HT9,M9P,and MT9)with safety switches.In vitro experiments confirmed that the H9P/M9P cell line can rapidly and specifically respond to CID-induced apoptosis,within 2 hours,and CID resulted in no killing effect on differentiated cells such as neural stem cells and wild-type pluripotent stem cells.In vivo experiments showed that H9P cells without administration of CID,a large number of cells remained undifferentiated two weeks after transplantation,while pre-administration,simultaneous administration,and early administration of CID after transplantation of H9P/M9P cells could effectively inhibit teratoma formation in vivo.The results above suggest that the OCT4-iCasp9 can serve as a direct,rapid,and safe strategy to overcome tumorigenesis during pluripotent stem cell therapy.In summary,for the first time,we have constructed a novel base editor TaC9-ABE resulting in no Cas9-dependent off-targets,as well as the pluripotent stem cell lines carrying the inducible suicide switch(OCT4-iCasp9),thereby improving their application safety.Both are expected to play an important role in future clinical practice.