Application of RNA-targeted CRISPR/CasRx system in mouse spermatogonia cell line GC1-spg

Objective To investigate the application of CRISPR/CasRx mediated gene knockdown at the RNA level in mouse spermatogonia cell line GC1-spg. Methods The EF1a core promoter. CasRx.SV40.U6.DRs expression plasmid (CasRx-gRNA) and three fluorescent protein of EF1a.EGFP, EF1a.mCherry, EF1a.tdToamto expression plasmids were constructed by homologous recombination. Three fluorescent protein expression plasmids and CasRx-gRNA expression plasmids were transiently transfected into human embryonic kidney cell line HEK-293T, and the knockdown of exogenous gene (the fluorescent proteins,EGFP and mCherry) was used to detect by fluorescence intensity and Western blot. Additionally, the expression levels of endogenous gene (mRNA and lncRNA) were measured by q-PCR in HEK-293T cells transfected with CasRx-gRNA. The exogenous gene egfp, mCherry and CasRx-gRNA expression plasmids were transiently transfected into the mouse spermatogonia cell line GC1, and the knockdown efficiency of exogenous gene (the fluorescent proteins) was detected by the above method. Results CasRx-gRNA and three kinds of fluorescent protein expression plasmids were constructed successfully. After CRISPR/CasRx mediated RNA interference of exogenous genes (egfp, mCherry) and endogenous genes (mRNA, lncRNA) in HEK-293T cells, the expression level was significantly reduced (P＜0.01). The CRISPR/CasRx system was verified in the mouse spermatogonia cell line GC1, which could effectively and specifically knock down exogenous genes egfp and mCherry. Conclusions CRISPR/CasRx system exerts an effective and specific knock-down function in GC1-spg, providing a new gene silencing tool potentially used in the study of male reproduction.