CRISPR/Cas9-mediated double knockout of SRPK1 and SRPK2 in a nasopharyngeal carcinoma cell line.

ABSTRACT

BACKGROUND: Serine-arginine protein kinase (SRPK) is a regulator of alternative splicing events via phosphorylation of splicing factor proteins. Oncogenic roles of SRPK1 and SRPK2 have been reported in various types of cancer. To date, only SRPK1/2 specific inhibitors and small interfering RNA (siRNA) have been used for halting their function momentarily; however, there is no attempt to generate SRPK1/2 stable knockout cancer cells as a tool to investigate their roles in tumorigenesis. AIM: Our objective is therefore to establish a nasopharyngeal carcinoma (NPC) cell line with stable SRPK1 or SRPK2 knockout and SRPK1/2 double knockout as a model to investigate their potential roles in NPC. METHODS AND RESULTS: CNE1 was selected as a representative of NPC cell lines to create single and double knockout of SRPK1/2 proteins. SRPK1/2 KO plasmid with cas9, green fluorescent protein (GFP), and gRNA expression was cotransfected with SRPK1/2 homology-directed repair (HDR) plasmid containing puromycin resistance, red fluorescent protein (RFP), and 5' and 3' arm sequence for homologous recombination to CNE1 cells. The transfected CNE1 cells with GFP and RFP expression were sorted through fluorescence-activated cell sorting for further treatment with puromycin containing medium. This step generated stable single knockout of SRPK1 and SRPK2. The SRPK2 knockout NPC cells were used as a precursor for double knockout generation via transfection with Cre plasmid for excision of inserted material to generate puromycin-sensitive SRPK2 knockout clone. The puromycin-sensitive SRPK2 knockout cells were transfected with SRPK1 KO/HDR plasmid and treated with puromycin-containing medium. The puromycin-resistant cells of SRPK1/2 stable double knockout were expanded, and the corresponding protein expression was confirmed by western immunoblotting analysis. CONCLUSION: Single and double knockout of SRPK1/2 were established using clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) system in an NPC cell line as a model for investigation of their splicing mechanism in NPC.