Base Editor Mediates Site-specific Mutation Of Multiple Fetal Genes In Tibetan Sheep Cells And Improves Editing Efficiency

Targeted modification of DNA sequence through gene editing technology is one of the important methods for organisms to acquire new characteristics or functions.Based on the CRISPR/Cas9 system,the derived single base editing system gradually excelled in the field of gene target modification.The nickase Cas9(n Cas9)component kept the double strand of DNA from breaking.It greatly reduces the Insertion and Deletion(Indels)of DNA fragments during editing.And the single base editing system is favored in the treatment of biological genetic diseases because of its excellent biological safety.The single base editing system has gradually become an important molecular breeding method for improving economic characters in animal husbandry.Oula-type Tibetan sheep is a unique breed of sheep in China,which has the advantages of outstanding growth performance,rich meat nutrition and high economic value.To build Tibetan sheep industry is of great significance to help the sustainable development of characteristic agriculture and animal husbandry in Qinghai Province.However,the low production efficiency of Tibetan sheep has become the biggest obstacle to the development of its industry,and it is difficult to rapidly increase the number of lambs by traditional breeding methods.Fec B and GDF9 have been proved to be the main genes of multiple reproduction in Tibetan sheep.There are A746 G mutations in the coding region of Fec B gene,and G260 A,G721A and G1184 T mutations in the coding region of GDF9 gene,which may improve the reproductive traits and reproductive performance of Tibetan sheep.The aim of this experiment is to use the single base editing system to achieve the target site mutation of Fec B and GDF9 genes in Tibetan sheep fibroblasts,and introduce small molecule compounds that promote the Homology directedrepair(HDR)DNA repair pathway.It is expected to solve the problem of low efficiency of target site editing in livestock cells.The detailed test process and results are as follows:(1)Single guide RNA(sg RNA)was designed according to the gene sequence of Fec B target site,and the sg RNA was connected to epi-ABEmax vector;Sgrnas were designed for G1,G4 and G8 target sites of GDF9 gene,and then each sg RNA was connected to epi-BE4 max vector.Subsequently,the vectors were transferred into Tibetan sheep fibroblasts by electroporation.After drug screening,the genome of positive cells was extracted,and then the base substitution of target sites was determined by Sanger sequencing,and the editing efficiency was tested by T-A cloning.Finally,the potential off-target sites of Fec B gene were predicted and their gene sequences were detected.The results showed that epi-ABEmax could mediate the base substitution from A to G of Fec B target sites in Tibetan sheep fibroblasts,and the editing efficiency of this site was 39.13%.epi-BE4 max could mediate G to A base substitution at G1,G4 and G8 target sites of GDF9 gene in Tibetan sheep fibroblasts,with editing efficiency of 10.53%,26.67% and 8%,respectively.(2)In the process of Fec B gene target mutation mediated by epi-ABEmax editor,Nexturastat A,Ricolinostat,RS-1 and SB-505124 were added respectively.To detect whether these small molecules can promote the editing efficiency of Fec B gene target sites in Tibetan sheep cells and 293 T cells.The editing efficiency was tested by T-A cloning and ampltron sequencing.It was found that Nexturastat A and RS-1 small molecule compounds could improve the editing efficiency of Fec B gene target sites in Tibetan sheep fibroblasts,and the base substitution frequency of Nexturastat A and RS-1 increased by 0.61 and 0.39 times,respectively.Nexturastat A also improved the editing efficiency of Fec B gene target sites in239 T cells,with a 0.33-fold increase in base substitution frequency.(3)In the process of GDF9 gene target mutation mediated by the epi-BE4 max editor,Nexturastat A,Ricolinostat,RS-1 and SB-505124 were added respectively,and whether these small molecules could promote the editing efficiency of GDF9 gene target site in Tibetan sheep cells was detected.The editing efficiency was tested by T-A cloning and ampltron sequencing,and it was found that Nexturastat A and SB-505124 could improve the editing efficiency of G1 target site of GDF9 gene in Tibetan sheep fibroblasts,and the base substitution frequency was increased by 0.48 times and 0.34 times,respectively.Nexturastat A and SB-505124 can also improve the editing efficiency of GDF9 gene G4 target site in Tibetan sheep fibroblasts,and the base substitution frequency of Nexturastat A and SB-505124 can be increased by 0.37 and 0.36 times,respectively.In conclusion,in this study,the single base editing system was used to realize the sitedirected mutation of Fec B and GDF9 multiple fetal genes at the cellular level of Tibetan sheep.Sanger sequencing,T-A cloning and deep targeted sequencing were used to detect the editing efficiency and determine the type of gene editing.Four small molecule compounds,Nexturastat A,Ricolinostat,RS-1 and SB-505124,were also added in the editing process,and the effect was evaluated by ampltron sequencing.It is the first time that Nexturastat A and RS-1 can improve the editing efficiency of Fec B gene target sites in Tibetan sheep cells.Nexturastat A and SB-505124 can improve the editing efficiency of GDF9 gene target sites in Tibetan sheep cells.