Construction,Application And Optimizationof An Enrichment System For CRISPR/Cas9-mediated Precision Gene Editing In Mammalian Cells

The precision genome editing allows scientists to directly change the gene sequences for investigating gene function and for developing safe and highly precise gene therapy approaches.The CRISPR/Cas9 system,which utilizes a Cas9 nuclease protein that complexes with a single guide RNA(sgRNA),is empowering researchers to perform genome editing more efficiently by inducing a site-specific DNA double strand break(DSB)within a targeted genome.The DSB is mainly repaired by two nonhomologous end joining(NHEJ)and homology directed repair(HDR)mechanism.Contrast to NHEJ repair which often resulting in insertion or deletion(indels)mutations at the DSB,HDR utilizes an exogenous DNA donor template homologous to region surrounding the DSB to repair DNA in a precise manner.The core of precision genome editing is based on the HDR mechanism.However,in mammalian genome,the efficiency of HDR repair is much lower than that of NHEJ repair,which limits the use of precision editing in mammalian cells.To improve the efficiency of precision genome editing,researchers have employed the different strategies,including to inhibit NHEJ pathway,activate HDR pathway and alter cell-cycle parameters.All of these approaches improved the efficiency of HDR to some extent.In addition,isolating genetical modified cells from all treated cell population is also technically challenging and time consuming because of the low HDR efficiency.The initial enrichment methods aim for either selecting transfection-positive cells or selecting the cells bearing the nuclease cleavage events.However,there is no report on a strategy for screening of CRISPR/Cas9 and HDR-mediated precision edited cells.In this study,we developed a universal surrogate reporter specific for enrichment of CRISPR/Cas9-mediated HDR,which we dubbed “HDR-USR”(Homology Directed Repair-based Universal Surrogate Reporter)system.This HDR-USR system works independently of other vectors.To use this system,we only need to co-transfect it with desired sgRNA and donor targeting an interested site into cells.After 2 days of transfection,we need to add puromycin into cell culture medium for 5 days of drug selection,then we can efficiently enrich the HDR-mediated point mutation,fragment knock-in and fragment deletion.This HDR-USR system can be widely applied at multiple loci in various mammalian cells.The main results of our study are shown as follows:1.We designed an HDR-USR vector,the HDR-USR reporter vector contains a universal sgRNA expression cassette,a Cas9 gene expression cassette,a truncated puromycin resistant gene expression cassette which contains a universal sgRNA target sequence,and a puromycin resistant sequence without promoter and start codon used as an intro-molecular repair template.When HDR-USR was transfected into cells,the expressed Cas9 protein will be guided by the expressed sgRNA to cleave the truncated puromycin resistant gene expression cassettes,and then the cleaved puromycin will be repaired by the puromycin resistant sequence,resulting in puromycin resistant gene expression.2.For application in point mutation,we tested our HDR-USR system at 6 loci(EMX1,AAVS1,CCR5,VEGF,HPRT1,NUDT5 loci)in 4 different mammalian cell lines(HEK293T,U2 OS,Hela,and A375),one locus(COSMC)in 2 cell lines(B16 and CHO)and IGF2 locus in PK15 cell lines.Through enzyme detection and deep sequencing for amplicons,we detected the HDR efficiency of the enriched HDR-mediated precision point mutations up to 45.93%,and enhanced 20.7-fold compared with the no selection group.3.For application in precision dual-locus editing,we tested our HDR-USR system for simultaneously point mutation at EMX1 and AAVS1 loci in HEK293 T cells.Through picking cell clones and genotyping,we detected the dual-locus editing efficiency reached up to 42% compared to zero in the no selection group.4.For application in fragment knock-in,we tested our HDR-USR system at GAPDH locus in HEK293 T cells.Through flow cytometry analysis,we detected the eGFP knock-in efficiency reached up to 38.5% in HDR-USR group,and enhanced 14-fold compared with the no selection group.5.For application in fragment deletion,we tested our HDR-USR system at AAVS1 locus in HEK293 T cells.Through picking cell clones and genotyping,we detected the biallelic deletion efficiency reached up to 61% in HDR-USR group,and enhanced 35.9-fold compared with the no selection group.6.We investigated the effects of yeast Rad52(yRad52),Ad4E1B-E4orf6,SCR7,nocodazole,ssODN donor,linear or plasmid donors and found that they can further improve the HDR efficiency based on the enrichment by our HDR-USR system.In summary,the HDR-USR system provides a simple,robust and efficient surrogate reporter for enrichment of the cells with CRISPR/Cas9-induced HDR-mediated precision genome editing without perturbation of genome integrity,and it provides an effective way to promote the application of gene editing in basic functional research,precision medicine and animal genetic breeding.