HEK-293T cells with USP30 knockout: construction using the CRISPR/Cas9 system and effects on Senecavirus A replication

Abstract:

[Objective] The clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) system was employed to knock out the ubiquitin-specific protease 30 (USP30) gene from human embryonic kidney 293T (HEK-293T) cells, which established a cell model for studying the function of USP30 and the regulatory role of this protein in virus replication.[Methods] According to the sequence of the first exon in the overlapping region of different transcripts of USP30 in Ensemble, we designed two pairs of single guide RNAs (sgRNAs) and constructed the recombinant plasmids pX459-USP30-sgRNAs. The HEK-293T cells were transfected with pX459-USP30-sgRNA and treated with puromycin for the screening of transfection-positive cells, from which the monoclonal cells were screened out by limited dilution method. We then employed Western blotting and DNA sequencing to evaluate the knockout of USP30 and compare the replication of Senecavirus A (SVA) in the wild type and the cells with USP30 knockout. [Results] We confirmed that USP30 was knocked out from HEK-293T cells by Western blotting and DNA sequencing. Further experiments revealed that the replication level of SVA in the cells with USP30 knockout was significantly lower than that in the wild type, indicating a positive role of USP30 in SVA replication. [Conclusion] We successfully constructed the HEK-293T cells with USP30 knockout and confirmed that USP30 played a positive role in SVA replication. The constructed cell line provides a good cell model for further deciphering the mechanisms of USP30-associated immune response and SVA infection process and serves as a tool for studying the regulatory role of USP30 in viral replication.