Table 1

Primers for MSTN gene"

Table 2

sgRNA sequences for MSTN gene"

Fig.1

Map of the Cas9 vector targeting MSTN gene"

Fig.2

Schematic diagram of the vector-targeted knockout site for MSTN gene"

Table 3

Primers used for off-target site detection"

Table 4

crRNA and tracrRNA sequences"

Table 5

Different electroporation protocols"

Table 6

Specific primers for nested PCR"

Fig.3

Sequencing alignment results of MSTN gene exon 2 in Huaxi cattle"

Fig.4

Sanger sequencing chromatogram of Cas9 knockout vectorOrange letters represent the sgRNA sequences； Black letters represent the PX458 vector sequences"

Fig.5

Fluorescent expression of cells after vector electroporation observed under a fluorescence microscope"

Fig.6

Quantitative analysis of transfection efficiency by flow cytometryFITC represents fluorescence intensity；SSA represents cell count"

Fig.7

Comparison of transfection efficiencies of different vectors*， Significant difference （P<0.05）； **， Extremely significant difference （P<0.01）. The same as below"

Fig.8

Validation of MSTN protein expression after Cas9 vector knockoutA， Expression of MSTN protein in cells of CON and after vector editing detected by Western blotting； B， Relative expression of MSTN protein"

Fig.9

Mutational analysis of monoclonal MSTN knockout cell lines from single vectors①A and B， The mutation status of monoclonal cell clones targeting the MSTN gene exons 1 and 2 obtained through flow cytometric fluorescence sorting， respectively. ②The yellow square represents the PAM sequence； The blue letters are the sgRNA； The red letters are the mutant base； The green letters are the insertion bases. ③WT， Wild-type cells； Edited， Gene-edited cells； MUt， Mutant. ④Different symbols represent different types of mutations， the numbers indicate the quantity of bases inserted or deleted； Mut， Mutant； #， Missing bases； -， Deletion mutation； +， Insertional mutation； &， Multi-base mutations. The same as below"

Fig.10

Mutational analysis of monoclonal MSTN exon 2 knockout cell lines from dual-vectors"

Table 7

Monoclonal cell line screening statistics"

Fig.11

Information on off-target sites of Cas9 vectorA-F， Information about off-target sites for each vector； The red box is the target information corresponding to the vector； The white box indicates that the bases of the potential off-target site are consistent with the target bases， while the purple box indicates that the bases of the both are different"

Fig.12

Off-target sequencing results of Cas9 vector① A， Agarose gel electrophoresis results of PCR products for off-target efficiency detection； B， Sequencing information for off-target sites targeting exons 1 and 2， respectively. ②OF1-OF5 corresponds to five off-target sites of vector E1-1， OF6-OF10 corresponds to five off-target sites of vector E1-2， and so on"

Fig.13

Embryonic development under different electroporation conditionsValue with different letter superscripts mean significant difference （P<0.05）；While with the same letter superscripts mean no significant difference （P>0.05）"

Fig.14

Gene-edition of electroporated embryos① A， Cleavage embryos after electroporation； B， Embryos that have developed to blastocyst stage after electroporation； C and D， Nested PCR amplification results targeting exons 1 （1-20） and 2 （25-38） of MSTN gene，respectively； E and F， Sanger sequencing results of targeting MSTN gene exons 1 and 2 edited embryos，respectively. ②M， DNA Marker Ⅰ； 1-38， Edited blastocyst identifiers"