Preparation Of MSTN Gene Knockout Sheep By CRISPR/Cas9

Myostatin(MSTN)is the negative regulation of skeletal muscle cell growth factor,the gene's mutation could make individuals show the “double muscle” phenotype.This study used the CRISPR/Cas9 System to knockout sheep MSTN gene to provid germplasm innovation material for Sheep in the future.(1)The design and activity validation of CRISPR/Cas9-MSTN-g RNA : Selecting 10 targeted site from Sheep MSTN gene CDS,then building Cas9 / g RNA to four targeted site to,then getting the highest cutting efficiency(55%)for GDF8-g RNA-1 target site after verifing the activity in vitro.(2)Effects of sheep sperm sources on IVF : The cleavage rate of epididymis fresh sperm and frozen sperm ware 85.03% and 71.25% respectively,and epididymis fresh sperm's cleavage rate was significantly higher than frozen sperm(P<0.05).The blastocyst rates of epididymis fresh sperm and frozen sperm ware 48.74% and 31.76% respectively,and epididymis fresh sperm's blastocyst rate was significantly higher than frozen sperm's(P<0.05).(3)Effects of sperm-oocyte co-cuture time(18h?22h?24h?26h)on IVF : The cleavage rates of sperm-oocyte co-cuture time(18h?22h?24h?26h)on IVF ware 55.13%?83.06%?81.09%?73.91% respectively.The cleavage rates of sperm-oocyte co-cuture time for 22 h and 18 h ware significantly higher(P<0.01).22 h was significantly different to 26h(P<0.05),and not significantly different to 24h(P>0.05).The blastocyst rates of sperm-oocyte co-cuture time(18h?22h?24h?26h)on IVF ware 27.91%?44.66%?35.33%?28.23% respectively.The blastocyst rate of sperm-oocyte co-cuture time for 22 h was significantly different to the 18h?24h?26h(P<0.05).The results showed that 22 h was the better time for oocyte in vitro fertilization(IVF).(4)Effects of injection exogenous RNA time(9h?10h?11h?12h)on embryonic development : The cleavage rates of injection time(9h?10h?11h?12h)ware 42.95%?32.26%?20%?13.56% respectively.The cleavage rate of 9h was significantly different to 11h?12h(P<0.01),and significantly different to the 10h(P<0.05).The blastocyst rates of those(9h?10h?11h?12h)ware 37.50%?14.44%?0.87%?12.5% respectively.The blastocyst rate of 9h was significantly different to 10h?11h?12h(P<0.01).The results showed that 9h was better injection time for development.(5)Effects of injection exogenous RNA concentration on embryonic development : The cleavage rates of injection exogenous RNA concentration((1)(Cas9:50ng/ul,g RNA:25ng/u L)?(2)(Cas9:25ng/u L,g RNA:12.5ng/u L)?(3)(Cas9:12.5ng/u L,g RNA:6.25ng/u L))ware 41.67%?57.25%?59.09% respectively.The cleavage rate of injection exogenous RNA concentration for(2)was significantly different to(1)(P<0.05),not significantly different to the(3)(P>0.05).The blastocyst rates of injection exogenous RNA concentration((1)(Cas9:50ng/ul,g RNA:25ng/u L)?(2)(Cas9:25ng/u L,g RNA:12.5ng/u L)?(3)(Cas9:12.5ng/u L,g RNA:6.25ng/u L))ware 20%?37.97%?38.46% respectively.The blastocyst rate of injection exogenous RNA concentration for(2)was significantly different to(1)(P<0.01),and not significant differences to the(3)(P>0.05).(3)(Cas9:12.5ng/u L,g RNA:6.25ng/u L)was better injection concentration.(6)The cutting efficiency of g RNA-Cas9 for sheep embryo : The cutting efficiency of m RNAmix was 23.5% when injection concentration for Cas9:25 ng/ul;g RNA : 12.5 ng/ul were used at 9h.(7)After blastocyst transplant,MSTN gene knockout sheep offspring was born in Oct 2016.