Abstract: The present disclosure discloses a PE-STOP gene editing system and gene knockout method and application. The gene editing system includes a prime editor protein, a pegRNA targeting a target site, and a matching nicking sgRNA for cleaving. The prime editor protein is selected from a PEmax protein, and an amino acid sequence of the PEmax is shown in SEQ ID NO. 1. The PE-STOP gene editing system can perform base replacement of a target gene sequence and introduce a termination codon in advance, thereby efficiently achieving the knockout of the target gene. The gene knockout method provided by the present disclosure has higher genotypic purity of the editing product and lower off-target activity, as well as higher genome coverage depth, therefore it has broad application prospects.