Abstract: The present invention discloses a CRISPR/LpCas9 gene editing system and application thereof, the CRISPR/LpCas9 gene editing system includes a complex of LpCas9 protein and sgRNA, which can accurately locate a target DNA sequence and cleave DNA double strands. The LpCas9 protein has an amino acid sequence shown in SEQ ID NO:1; and the sgRNA has a nucleotide sequence shown in SEQ ID NO:2, or a modified sgRNA sequence based on SEQ ID NO: 2. The present invention can effectively solve the problems of heterologous codon bias and cytotoxicity in the application of SpCas9 in Lactobacillus paracasei. The gene editing is performed in cells or in vitro by introducing artificially designed CRISPR RNA and a repair template, which solves the problem of low electroporation efficiency caused by the strains' characteristics or relatively large carried plasmids, and has broad application prospects in the field of gene editing.