Abstract: Objective To target knockout the exon51 of DMD gene in HEK293T cells using the CRISPR/Cas9 system. Methods Design the target sequences of sgRNA and clone them into plasmid PX459 respectively; transfer these plasmids into HEK293T cell and extract the total genome DNA; test the activity of sgRNAs with surveyor assay, choose the most efficient one in each end; construct plasmid PX459-2sgRNA and transfer it into HEK293T cells; check whether the exon51 has been knocked known with PCR and T vector sequencing. Results 50% of HEK293T cells’ DMD gene exon51 were knocked out, showing a high gene editing efficiency. Conclusion Successfully establish a platform to target knockout the exon51 of DMD gene, provide an important experimental basis for the treatment of DMD and other genetic diseases.

Key words: CRISPR/Cas9, DMD gene, single vector plasmid, HEK293T cell, exon knockout