Knocking-out extra domain A alternative splice fragment of fibronectin using a clustered regularly interspaced short palindromic repeats/associated proteins 9 system / 中华口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of the fibronectin extra domain A on the aggressiveness of salivary adenoid cystic carcinoma (SACC) cells, via the clustered regularly interspaced short palindromic repeats (CRISPR)/ associated proteins (Cas) system.</p><p><b>METHODS</b>One sgRNA was designed to target the upstream of the genome sequences of extra domain A(EDA) exon and the downstream. Then the sgRNA was linked into plasmid PX-330 and transfected into SACC-83 cells. PCR and DNA sequence were used to testify the knockout cells, and the monoclones of EDA absent SACC cells were selected (A+C-2, A+C-6, B+C-10). CCK-8 cell proliferation and invasion was then tested in control group and the experimental group.</p><p><b>RESULTS</b>The sgRNA was successfully linked into PX-330 plasmid. Part of adenoid cystic carcinoma cells' SACC-83 genomic EDA exon was knocked out, and the knockdown efficiency was above 70%, but the total amount of fibronectin did not change significantly. Three monoclones of EDA absent SACC- 83 cells were successfully selected with diminished migration and proliferation.</p><p><b>CONCLUSIONS</b>The CRISPR/Cas9 system was a simplified system with relatively high knockout efficiency and EDA knockout could inhibiting SACC cell's mobility and invasiveness.</p>