Construction of microRNA-155 gene knockout mouse model using CRISPR/Cas9 technique

Abstract

Objective To explore for the construction of microRNA-155 (miR-155) gene knockout mouse model by using CRISPR/Cas9 technique. Methods Healthy C57BL/6J mice were selected. First, the target sequence of mouse genome was amplified and sequenced to identify the miR-155 sequence and loci. Then the Cas9 vector (Cas9 RNA) and targeting vector single guide RNA (sgRNA) were designed and constructed. After in-vitro transcription, Cas9 RNA and sgRNA were injected into the mouse fertilized eggs which were cultured in vitro. The qualified embryos were transplanted into the fallopian tubes of the surrogate mice which then gave birth to the F0 generation mice. The gene knockout of F0 generation mice was examined by the genotyping. Thereby, knockout mice model was established and bred. Results Eighteen mice of F0 generation were obtained by CRISPR/Cas9 technique. Mouse No.4 presented with deleted base segments in single-stranded DNA and stably passagable genotype. After reproduction, three F1-generation heterozygous mice were obtained. The heterozygous mice were mated to obtain F2 homozygous knockout mice. Electrophoresis and gene-sequencing confirmed deletion of 114-bp base sequence in the off-springs, suggesting successful knockout of miR-155 gene. The miR-155 gene knockout mice could reproduce normally in clean-grade animal breeding environments. Conclusion CRISPR/Cas9 technique can be used for quick and effective construction of miR-155 gene knockout mice models. Key words: MicroRNA-155; CRISPR/Cas9; Models, animal; Mice; Gene knockout