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

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.

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.

Gene deletion with acceptable efficiency (38%, p<0.05) successfully was confirmed by sequencing and PCR analysis.

Our result showed that the CRISPER/Cas9 technique is an effective knockout method in mouse zygotes, potentially producing disease animal models.