RNA-Seq of Flp-In T-REx 293, two clones of CRISPR-Cas9 mediated knockout of CASC3 and further CASC3 depletion via small interfering RNAs

We report two knockouts of the CASC3 (Barentsz, MLN51) gene and further depletion of gene expression with small interfering RNAs (siRNAs). CASC3 is a component of the exon-junction complex (EJC) which is deposited upstream of splice junctions on the mRNA. The EJC core and peripheral interacting factors are involved in RNA splicing, export, translation and nonsense-mediated decay. Contrasting to the other factors that form the exon-junction complex, CASC3 individual role on these processes was relatively poorly understood in the literature. To further elucidate the role of CASC3 in these processes we have established 2 different CRISPR-Cas9 genetic knockouts (KO) of CASC3. We extracted the total RNA by using the NucleoSpin RNA Plus kit (Macherey-Nagel), and performed ribosomal depletion and strand-specific library preparation with the TruSeq Stranded Total RNA protocol with Ribo-Zero Gold. Sequencing of the KO clones as well as the KO clones treated with CASC3 siRNA was carried out with the Illumina NovaSeq6000 sequencer with 2×100bp, targeting approximately 50 million read-pairs per sample. By studying the RNA-Seq of the 4 conditions, one with minimal CASC3 expression (condition H) or and three with complete depletion of the protein expression (conditions H-KD, T, T-KD), we observed the up-regulation of known and novel nonsense mediated decay substrates, thus establishing CASC3 as a critical factor for efficient nonsense mediated decay of certain transcripts.