The invention discloses a method for constructing high-quality porcine nuclear transplantation donor cells with high lean meat percentage, fast growth, high fertility and resistance to series epidemic diseases and application thereof. A CRISPR/Cas9 system for editing five genes of porcine MSTN, SST, INHA, CD163 and pAPN comprises a Cas9 expression vector and gRNA expression vectors respectively aiming at the porcine MSTN, SST, INHA, CD163 and pAPN genes; and the plasmid complete sequence of the Cas9 expression vector is shown as SEQ ID NO. 2. The corresponding gRNA expression vectors are respectively designed aiming at different target points of the MSTN, SST, INHA, CD163 and pAPN genes, and gRNA with higher editing efficiency and expression vectors thereof are obtained through screening. The gene editing is performed by matching with the modified Cas9 high-efficiency expression vector, and the editing efficiency is obviously improved compared with that of the original vector.