Abstract: To study the function of miR-155 in porcine alveolar macrophage cell line（3D4/21）,miR-155 knockout cells were constructed using CRISPR/Cas9 technology. Firstly,4 prime series specific to sgRNAs for targeting miR-155 were designed using sgRNA-cas9 program. Then,sgRNA expression vectors were constructed and co-transfected with pEGFP-C1-cas9 vector into 3D4/21 cells. Next,the cells expressing green fluorescent protein（GFP）were enriched and selected using the FACS-sorting strategy. Finally,T7E I cleavage assay,Sanger sequencing,real-time quantitative PCR（RT-qPCR）and Western blot were used to detect the efficiency of the knockout. We found that miR-155’s genomic sequence was edited using 4 different sgRNAs,while the highest efficiency of knockout reached by sgRNA39,up to 42% after T7EI enzyme digestion and FACS-sorting. Sanger sequencing demonstrated that 31 nucleotides of sgRNA39 cell line were knocked out. The RT-qPCR result showed that the expression levels of miR-155-5p/3p significantly decreased. The Western blot result indicated that the protein expression level of target gene SHIP1 in miR-155 significantly increased. To sum up,the successful establishment of the miR-155 knockout cell line provides cell model for further studying the regulation function of miR-155 in macrophage cells.

Key words: CRISPR/Cas9, miR-155, knockout, porcine alveolar macrophage cell line