A Preliminary Study On The Effect Of CRISPR/Cas9-mediated Targeting Of Mouse Rbmy Gene On The Sex Ratio

The technology of sex control is a kind of technology which can be used to intervene in the process of animal reproduction,which makes the female animals produce a kind of technology which is suitable for the offspring of a specific sex.Many techniques such as flow cytometry sperm separation technology,sex related gene silencing and sex identification of early embryos,animal somatic cell cloning technology can have a certain effect on the control of different species in a certain extent,but there is not a large-scale sex control technology can be used in different mammalian animal.Therefore,it is very important to find an effective,convenient and fast method for sex control.The study of CRISPR/ Cas9 system targeting Y Chromosome-based m?lti-copy Rbmy gene by transgenic to study its effects on mouse sex ratio,provide the basis for the study of livestock sex control technology.In this study,6 target sites were selected for the Rbmy gene,and the best sgRNA4 and sgRNA6 were optimized by sgRNA mediated Cas9 protein cleavage in vitro.Three sgRNA and Cas9 co-expression vectors which targeting mouse Rbmy gene were constructed by using the Spermatogenesis specific Protamine1 promoter,the embryonic development specific Trim43 promoter and systemic expression promoter CMV.The targeting vector reg?lated by CMV(vector one)was transfected into MLTC-1 cells derived from male mouse,to study its lethal efficiency.In addition,the transgenic mouse were produced by microinjection of the linearized plasmid into the fertilized eggs,and the sex ratio of the fertilized eggs to blastocysts and the development to the birth was studied.The res?lts showed that: compared with vector transfected group and negative control group,transfected MLTC-1 cells after 12h-36 h,green fluorescent cells ratio of two groups had no difference,indicating that the two groups had no significant difference in transfection efficiency,but after transfection of 42h-52 h,a vector transfection group green fluorescent cell ratio was significantly less than two vector transfection group(negative control),and flow cytometry res?lts showed that after transfection of 42 h,a vector transfection group green fluorescence efficiency was 16.2%,lower than vector two transfection group(negative control)of 27.8%,Which indicate a vector have the capable of cutting Y chromosome and killing MLTC-1 cells.The res?lts of sex determination of blastocysts showed that there was no significant effect on the sex ratio of embryo development to blastocyst(p>0.05).The res?lts of sex identification in born mouse displayed that a carrier injection had no significant effect on the sex ratio of embryo development to birth(p>0.05).In addition,thro?gh the identification of the 68 F0 generation mouse that were injected with embryonic development,only 7(2 male and 5 female)transgenic mouse,5 female transgenic mouse were then bred,46 mouse of F1 generation,the male and female ratio of 24/22,the sex ratio has no significant deviation.