〔Abstract〕 To investigate the role and mechanism of Lck/Yes-related novel protein tyrosine kinase （Lyn）on lipopolysaccharide（LPS）-induced M 1-type polarization of macrophage. Methods The LentiCRISPR- V2 plasmid was digested with the restriction endonuclease BSMBI-V2，and the digested DNA fragments were recov- ered. The digested plasmid was ligated with Lyn-sgRNA using T4 ligase to generate the Lenti-Lyn-gRNA lentivi- rus. THP-1 cells were infected with the Lenti-Lyn-gRNA lentivirus to obtain a stable cell line with Lyn knockout， and a monoclonal THP-1 cell line with complete Lyn knockout（Lyn⁻/⁻ ) was established subsequently. Wild-type Lyn（LynWT）and Lyn⁻/⁻ THP-1 cells were induced with 100 ng/mL phorbol myristate acetate（PMA）for 48 h to dif- ferentiate into M0 macrophages，which were further polarized into M 1 macrophages by stimulation with 100 ng/mL LPS for 24 h. Quantitative real-time polymerase chain reaction（qPCR）was performed to detect the expression of M0 macrophage markers，including integrin αM（CD11b），macrophage antigen（CD68），and monocyte differen- tiation antigen（ CD14）. The expression of Lyn in M 1 macrophages differentiated from wild-type THP-1 cells （LynWT-M1）was measured by qPCR，and the ratio of phosphorylated Lyn to total Lyn（P-Lyn/Lyn）in LynWT-M1 cells was determined by Western blot. In M 1 macrophages differentiated from Lyn-knockout THP-1 cells （Lyn⁻/⁻-M1），qPCR was used to detect the mRNA expression of inducible nitric oxide synthase（iNOS），interleu- kin-6（IL-6），and chemokine（C-X-C motif）ligand 10（CXCL-10）. Western blot was conducted to assess the pro- tein expression of iNOS，as well as the protein levels of molecules related to the Janus kinase 1（JAK1）-signal transducer and activator of transcription 1（STAT1）signaling pathway，including JAK1，phosphorylated JAK1（P- JAK1）， STAT1，and phosphorylated STAT1（P-STAT1）. Additionally，the expression of the M 1 macrophage marker cluster of differentiation 80（CD80）was analyzed by flow cytometry. Results The Lyn-/- monoclonal cell line was successfully constructed. The expression of CD11b was significantly elevated in Lyn-/- M0 macrophages， and the differentiation of M 1 macrophages was successful. Knockdown of Lyn inhibited mRNA expression of iNOS， IL⁃6，CXCL⁃10，protein expression of iNOS and CD80 expression in M 1 macrophages（P<0. 05）. Western blot as- say showed that Lyn knockdown inhibited protein expression of JAK1 and P-STAT1（P<0. 01）. Conclusion After CRISPR/Cas9-mediated Lyn knockout，the expression levels of JAK1 and P-STAT1，the key molecules in the JAK/ STAT signaling pathway of M 1 macrophages，are significantly downregulated；concomitantly，the expression of M 1 macrophage-specific secretory factors（iNOS，IL ⁃ 6，CXCL ⁃ 10 mRNA）and CD80 is also downregulated，which may be achieved via targeted regulation of the JAK1/P-STAT1-mediated JAK/STAT signaling pathway.