Delivering CRISPR/Cas9 With PiggyBac And Episome Systems

The 3rd generation genome editing tools,especially CRISPR/Cas9,have been widely used in basic research,technology development and biomedical research.Cas9 nuclease and sgRNA,two necessary components of CRISPR/Cas9 technology,are often used as in vitro transcribed Cas9 mRNA and sgRNA complex,RNP complex of Cas9 protein and in vitro transcribed sgRNA,and vectors containing Cas9 and sgRNA expression elements.The former two complexes are relatively safer than vectors but suffered from complicated process of preparation of both components thus precluding their further applications.As for commonly used plasmid vectors,they show low gene editing efficiency and are not convenient for integration detection.To solve the aforementioned problems,this study combined piggyBac and episome system with CRISPR/Cas9 with the expectation of advancing vector system for more applications.We constructed piggyBac-based hCas9 expression system upon DOX induction(hereafter called PB-iCas9 system).Firstly,we testified PB-iCas9 system for single gene editing in mouse embryonic stem cells(mESCs)against Cdkn2a,Pten and Trp53 three tumor suppressor genes(TSGs).Next,we co-injected PB-iCas9 vectors(targeting Cdkn2a,Pten and Trp53)and PB-NRASG12V IRESEGFP through tail intravenous injection.The functionality of PB-iCas9 system was confirmed by inducing liver tumors efficiently thus suggesting its potential use for building liver tumor models in vivo.The gene editing ability of PB-iCas9 system was confirmed by both in vitro and in vivo experiments.Finally,we located PB-iCas9 integration sites by inverse PCR and verified that Cas9 expression was under tight control of DOX by qPCR.Subsequent expression of PBase efficiently excised exogenous fragment from mouse genome.Analyses of mESCs with PB-iCas9 removal showed normal karyotypes and pluripotency as wildtype cells and suggested the possibility of using PB-iCas9 system for deriving vector-free mESCs to create transgenic animals.We also constructed oriP/EBNA1-based episome vector system(hereafter called oe-Cas9 system).We first introduced reprogramming factors and tdTomato into mouse embryonic fibroblast cells to derive mouse induced pluripotent stem cell lines with single tdTomato integration.After delivering oe-Cas9 system into miPSC,we confirmed its gene editing ability for targeting exogenous reporter gene.We further verified dual sgRNA-mediated CCR5 deletion and multiplex gene editing of TET familiy by oe-Cas9 system.Finally,we derived vector-free gene modified mammalian cells at both DNA and protein levels.In conclusion,both PB-iCas9 system and oe-Cas9 system could mediate efficient gene editing and might be candidate systems to derive vector or transgene-free cell lines.Considering the ease for use property of both systems,they could be useful tools for generating tumor models,gene modified cell lines or animals.