Verification of ADAR1 Knockout In an ADAR1/APOBEC1 Double Knockout

RNA editing causes a change in the RNA sequence without causing a change in the genomic DNA. The most common type of editing, adenosine to inosine (A-to-I), accounts for 90% of editing in the human transcriptome and is mediated by adenosine deaminases acting on RNA (ADARs). These editing events have been shown to be essential to development. This project focuseds on the development of a cell line with a dysfunctional product for the ADAR1 and APOBEC1 proteins. In order to validate the absence of ADAR1, a Western blot was performed on single-cell subclones of the APOBEC1 KO RAW 264.7 cells that had previously been edited via CRISPR/Cas9 to cause a deletion in the ADAR1 gene. After the Western blot showed no detectable ADAR1 product, four subclones were selected for PCR, cloning, and transformation into E. coli. After overnight incubation, 20 colonies from each subclone were chosen for a colony-PCR. Those colonies which contained the correct size insert were sent for Sanger sequencing. The Sanger sequencing showed a successful deletion in the ADAR1 gene of all 80 colonies; however, three sequences came back for the ADAR1 gene when there should only be two. This could be due to ADAR1 potentially being multiallelic, the subclone not being a single cell clone, or a mutation in the original RAW 264.7 cells. Despite the 3 sequences, it appears that ADAR1 has been successfully knocked-out in these cells