Validate The Activity Of Nucleases And Enrich The Positive Cells Induced By Artificial Nucleases

The genome engineering technology has changed a lot in the past thirty years. The technology has developed from sample homologous recombination to make double strand breaks(DSBs) at selected genome sites, which improved the genome engineering efficiency significantly. The artificial targeted genomic engineering technology has evolved from zinc finger nucleases(ZFNs) and over transcription activator-like effector nucleases(TALENs) to recent fully developmental clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas) systems, these engineering tools play an important role in gene modification in cell level, gene function research and transgenetic animal production in animal model level and gene therapy for human. Meanwhile, during the whole process of genome editing methods development, all these three systems have their own shortage, except for the complex screening process for zinc finger, high skill needed for TALE assembling and PAM dependent for CRISPR/Cas system, there is still some problems for the genome editing efficiency. Based on above issues, we keep trying to establish some reporter systems to validate the efficiency of artificial nuclease platforms and improve their editing ability in the genome.In this research, we developed a high sensitive reporter system to validate the efficiency of nucleases in both yeast and mammalian cells. We designed a yeast-based ZFN screening and validation system by combining Gal4 transcription factors and single strand annealing(SSA) DNA repair pathway; in the mammalian cells, we constructed a dual-reporter SSA-RPG surrogate system to check the nucleases cutting ability and used for efficient enrichment of genetically modified cells; at the same time, we did some effects to improve the nuclease editing ability and compared different CRISPR/Cas system platforms designed by different labs. Following are the main results in this study:1. We designed a ZFN screening and validation system by adding ZFNs binding sites flanked by 30 bp direct repeats sequence in the middle of a Gal4 transcription factor in yeast, disrupting the open reading frame(ORF) of the Gal4 gene. The express of the ZFNs can cause a double stranded break at their binding sites, which promotes the DNA repair system to restore the expression of functional Gal gene via single strand annealing. Gal4 transcription factor expression leads to activation of following three reporter genes in AH109 yeast strain. In order to reduce the occur of spontaneous homologous recombination, which would result in false positive results, a series of direct repeats from 10 bp to 164 bp with 10 bp increase each flanking the ZFNs binding site were constructed. The results shown that 30 bp was the minimum length required for ZFNs induced single strand annealing, which also can almost eliminate spontaneous homologous recombination. Using 30 bp direct repeated homologous arms, ZFNs induced homologous recombination was efficient, while false positive was minimized. Thus, we found a simple, robust and sensitive ZFNs screening and activity verification system in yeast.2. Since isolate genetically modified mammalian cells generated by artificial nucleases are challenging, because they are phenotypically indistinguishable from other parental cells. To efficiently select genetically modified cells, we constructed two dual-reporter surrogate systems, denominated as NHEJ-RPG and SSA-RPG system based on their repair mechanisms. Repair efficiency comparing of these two reporter systems were did using two different nuclease platforms. In both CRISPR/Cas9- and ZFNs-induced double strand break repair studies, we found the sensitivity and efficiency of SSA-RPG reporter system with direct repeats length more than 200 bp were pretty higher than the NHEJ-RPG one. Through utilizing the SSA-RPG system, we got the enrichment for genome editing in several genomic loci with 6.3- to 34.8-fold improvement than non-selected cells. In short, the SSA-RPG reporter system can be utilized for designed nucleases activity validation and efficiently enrichment for genetically modified positive cells. Meanwhile, our system could be used by puromycin selection or FACS sorting according to different lab conditions.3. By multiple site-directed mutagenesis of Fok1 domain, we introduced Sharkey-ELD and Sharkey-KKR mutations on each side of ZFNs, under this condition only different FokI variants can form hetero-dimers which could reduce the off-target affects and improve the modification ability.All in all, in our study we constructed a sample and robust nucleases activity validation reporter system for both yeast and mammalian cells, meanwhile we also can use this reporter system for efficiently enrichment of genetically modified positive cells, and in turn lays the foundation of animal genetic modification and breeding using targeting nucleases.