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Toll-like receptor 2 (TLR2) is a pattern recognition receptor which plays an important role in innate immune system. In humans it’s encoded by the TLR2 gene and also been designated as CD282. Using CRISPR/Cas9 gene editing technology, we have established a TLR2 mutant WAe001-A-64 cell line from the original embryonic stem cell line H1. It has adopted two biallelic deletions in exon 3 of TLR2 which resulted in a frame shift and early termination in the translation of TLR2. Moreover, WAe001-A-64 has maintained the normal karyotype, pluripotent phenotype, parental cell morphology and the ability to differentiate into three germ layers.