关键词: PDE10A, CRISPR/Cas9 , 稳定细胞株

Abstract: Objective To construct the CRISPR/Cas9 system of knocking out the PDE10A gene. Methods Three 20 bp sgRNAs targeting exon 6, exon 7 and exon 11 of PDE10A were designed, chemically synthesized, and then inserted into linearized plasmid, PX330. Next, the correct PX330-sgRNA plasmids were transfected into HEK293T cells after verification by Sanger sequencing. The targeting efficiency was detected by SURVEYOR assay and the nicked site was further detected by Sanger sequencing. Finally, the stable PDE10A knockout 293T cell lines were selected by the limiting dilution method. Results The target nucleotide sequences were successfully inserted into the expected sites of vector and sequences were correct. The targeted exon 7 sgRNA can successfully knock PDE10A, and the targeting efficiency was up to 31.4 %. Stable knockout PDE10A cell line was selected successfully which harbored 2 bp deletion. Conclusion The CRISPR/Cas9 system of knocking out the PDE10A gene was successfully constructed.

Key words: PDE10A, CRISPR/Cas9 system , Stable cell lines