CRISPR/Cas9-mediated Gene Modification In Xenopus Tropicalis

Objectives:Xenopus tropicalis,the only diploid species in the Xenopus genus,is an important research organism in functional genomics and biomedical.Dispite loss-of-function genomic editing for X.tropicalis has been well developed,lack of efficiency and feasible methods to insert an exogenous DNA sequence into the X.tropicalis genome is becoming a barrier for X.tropicalis-relevant research.The aim of this study is to develop a strategy,based on CRISPR/Cas9-induced DNA double-strand break(DSB)in intron region and donor plasmid design,to efficiently insert a tag into genome for target gene modification through homology-independent DNA repair mechanism.The strategy presented here provides a valuable avenue for establishing human diseases models and might be adapted to many other organisms.Methods:(1)Myosin heavy chain(Myh6),specific expression in X.tropicalis heart,was used as target gene,then designed 5 gRNAs in the last intron and the most efficient cutting site was selected.(2)Construction of donor 1 and donor 2 vectors: PCR-amplified a short genomic fragment bearing the target site(bait)was cloned into plasmids,which containing one-bait and two-bait.(3)Construction of donor 3 and donor 4 vectors: PCR amplified the sequence of 3' untranslated regions(UTR)of target gene into plasmids of(2)to substitute the sequence of SV40-poly A.(4)Donors plasmid,Cas9 mRNA,and single guide RNA(sgRNA)were coinjeceted into one-cell stage embryos.Then analysis of integration efficiency:(1)4-5 days post-injection GFP and m Cherry expression were examined and sorted out with a fluorescence stereomicroscope;(2)The tadpoles(about 3 to 5)were collected in each group for genomic DNA extraction and PCR amplicons were sequenced to detect both 3' and 5' integration junctions;(3)The remaining tadpoles in each group were raised to adulthood for germ-line transmission analysis.(5)Analysis of integration efficiency at cellular level by following the same stategy:(1)Three sgRNAs were designed to target the last intron of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)locus in human genome,and the most efficient cutting site was selected via p CMV-EGxx FP system.(2)Constructed 4 donors plasmids to carry a T2A-EGFP cassette flanked by one or two baits.Results:(1)A highly efficient cutting site in the last intron of target gene was selected and sequencing datas showed that the targeting efficiency is above 60%.(2)Upon injection,varying amounts of tadpoles in all four groups displayed fluorescence signals and the efficiency of targeted integration at an average of 8%.(3)Targeted integration efficiency of donor 1 group was higher than donor 2 group,donor 3 group was higher than donor 4 group,revealed that one-bait mediated targeted integration efficiency was higher than two-bait mediated.(4)Targeted integration efficiency of donor 3 group was higher than donor 1 group,donor 4 group was higher than donor 2 group,revealed that the target efficiency of the group containing 3'UTR sequences was higher than which not.(5)No GFP expression could be observed in the groups without sgRNA,this indicate that the sgRNA is necessary to trigger integration of the donor plasmid.(6)Successful targeted integration events were verified by PCR amplification of 3' and 5'junction of founders genomic and amplicons sequencing.(7)The majority of junction sequences showed indels(68/70,?97%),only two displayed perfect recombination(2/70,?3%).(8)Varied degrees of EGFP signals were observed in all groups upon taking the same strategy at cellular level,and stronger EGFP fluorescence were seen in control groups.Conclusions :(1)The concurrent cleavage strategy based on CRISPR/Cas9 in mediating targeted integration for X.tropicalis is feasible and effective.(2)Inserting exogenous gene into genomic loci and driven by endogenous promoters without disrupting the targeted gene by designing the target site in last intron.(3)Taking two-bait mediated targeted integration strategy can remove the plasmid backbone,but less efficiency.(4)Endogenous 3'UTR plays a vital regulatory role in the expression of exogenous gene,but the specific regulatory mechanisms need to be further studied.(5)Following the same strategy was unable to detect effective gene modification at celluar level.