Toll like receptor(TLR)3 is a pattern recognition receptor,which identifies double-stranded viral RNA.This receptor plays a crucial role in the innate immune response.The aim of this study was to create TLR3 knock-out mice using CRISPR-Cas9 technology.In this study,we designed sgRNAs targeting exon 4 of mouse TLR3.The sgRNA expression vector and linearized Cas9 plasmid were constructed and transcribed.We microinjected sgRNA and Cas9 mRNA into the cytoplasm of one-cell-stage mice embryos.The resultant embryos were genotyped using T7E1 digestion,polyacrylamide gel electrophoresis and sequencing.The homozygous(TLR3-/-)animals were screened by crossing in the F2 generation;the expression of TLR3 was analyzed at the RNA and protein level.Histological structures of the spleen and thymus in TLR3 knockout mice were analyzed by hematoxylin and eosin(HE)staining.The ratio of peripheral blood lymphocyte subsets,the cell ratios of CD86+,CD49+,CD8+/CD3+in the blood,the expression of TRIF,IFN-β,Mx,IL-6 and TNF-a in spleen were analyzed by automatic biochemistry analyzer,flow cytometry and qPCR before and after stimulation with poly I:C.The results show that animals heterozygous for the TLR3 13 bp deletion were obtained in the founder generation,after three generations we were able to confirm homologous deletion in the TLR3 1ocus.RT-PCR and Western Blot showed that the TLR3 gene in TLR3-/-mice was not expressed at the transcriptional and protein level.After polyI:C activation,the blood lymphocyte ratio in these animals changed.The CD86+,CD8+/CD3+cell ratios decreased,and the expression of TRIF,IFN-β,Mx,IL-6 and TNF-α in the spleen was also reduced.Taken together these results support the conclusion that a TLR3 knockout mouse model was successfully created.These animals showed a suppressed innate immune response making it an excellent model for the innate immune response and the pathogenic mechanisms employed by some infectious diseases.