Efficient Genome Editing In Buffalo MSTN By Zygote Electroporation Of CRISPR/Cas9-Mediated System

Buffalo is an important meat and husbandry using domestic animal.Based on these important economic values,scientists' improvement of buffalo traits has been under study.The development of CRISPR/Cas9 technology has made it possible to quickly and efficiently improve the performance of buffalo.Studies have shown that the MSTN(myostatin)gene regulates the growth of skeletal muscles.In this study,an electroporation tool and multiple regular gRNAs were designed based on CRISPR/Cas9 system.In combination with electroporation technique,Cas9 protein and multiple regular gRNAs were introduced into buffalo fertilized eggs to knock out the MSTN gene.The results of the study are as follows:1.Determination of optimal electroporation parameters on buffalo fertilized eggsThe electroporation technique was used to introduce EGFP mRNA into buffalo fertilized eggs,and the optimal electroporation parameters were investigated based on the fluorescence positive rate of the fertilized eggs and the subsequent development of the embryos.When the pulse time(2ms),number of pulses(3),and pulse interval(0.2s)were constant,the development rate of blastocyst in the 30V pulsed embryos was significantly higher than that in the 40V and 50V groups,and the positive rate of embryonic fluorescence was lower then 40V group but higher than 50V group(P<.0.05).When the pulse voltage(30v),number of pulses(4),and pulse interval(0.2s)were constant,the positive rate of embryonic fluorescence in the 4ms group was significantly higher than that in the 2ms group(P<0.05),but the blastocyst rate was different,not significant(P>0.05);When the electric pulse time is 6 ms,the damage to the fertilized egg is greater.When the pulse voltage(30v),pulse time(4ms),and pulse interval(0.2s)were constant,the blastocyst rate of the 4th group had no significant difference with the 3 times(P>0.05),but the positive rate of fluorescence was Higher than the third group(P<0.05).When the pulse voltage(30v),pulse time(4ms)and number of pulses(4 times)were fixed,the blastocyst development rate was significantly higher in the 0.2s pulse interval group than in the 0.3s group(P<0.05),and the positive rate of fluorescence was significantly higher than the 0.1s and 0.3s groups(P<0.05).2.Screening of effective gRNAs for CRISPR/Cas9 SystemUsing the CDS sequence of the third exon of buffalo MSTN gene as a template,five gRNAs were designed at intervals of 10-200 bp,and five corresponding RGS reporter vectors were constructed.The cas9 expression vector,gRNA expression vector,and RGS reporter vector were co-transfected into 293T cells,and the three most active gRNAs were selected by fluorescence observation and flow cytometry analysising.The editing efficiency is 37.4%for gRNAl,36.3%for gRNA2,37.1%for gRNA3.The three gRNAs screened were co-transfected with cas9 vector into buffalo fetal fibroblasts(BFF).The results showed that all three gRNAs had endogenous activity.3.Electroporation of CRISPR/Cas9 System to Knockout Buffalo Maturation MSTN GeneThe gRNA,cas9 protein and electroporation were mixed,and electrical pulses were applied to the buffalo fertilized eggs using optimal electroporation parameters.This study compared the effects of mixing a single gRNA with multiple gRNAs and found that a single gRNA caused deletions of small fragments only near the PAM site,whereas a 69 bp deletion occurred when three gRNAs were mixed,and the efficiency of the three gRNA editing was high,a single gRNA is lower.The above results indicate that the suitable electroporation parameters for buffalo fertilized eggs are pulse voltage 30V,pulse frequency 4 times,pulse time 4ms,and time interval 0.2s.Electroporation technology can efficiently introduce CRISPR/Cas9 system components into buffalo In the fertilized egg,combined with multiple regular gRNAs,a large fragment knockout of the target gene MSTN can be performed..