Efficient of Toll‐Like Receptor 4 Knockout in Mouse Zygotes by CRISPER/Cas9:

Abstract Background: Transgenic animals are genetically modified animals to create a specific trait that imitates an indication of pathogenesis in humans. Toll-like receptors (TLRs) are implicated in immune regulation of the female reproductive tract and, subsequently, infertility rate. This study produced Toll-like receptor 4 (Tlr4) knockout blastocysts with single-guide RNA targeting for Tlr4 by CRISPER/Cas9 technique. Materials and Methods: Web CRISPER design tools designed single-guide RNAs (sgRNAs) targeting Tlr4 gene were designed by web CRISPER design tools. Then, two strands of sgRNAs were cloned into a linearized vector for producing a gRNA-expressing eCAS9-GFP vector. The vector was then injected into the male pronucleus in the fertilized oocytes in vitro fertilization (IVF) and do polymerase chain reaction (PCR) and sequencing. Results: Gene deletion with acceptable efficiency (38%, p<0.05) successfully was confirmed by sequencing and PCR analysis. Conclusion: Our result showed that the CRISPER/Cas9 technique is an effective knockout method in mouse zygotes, potentially producing disease animal models.