CRISPR‐Cas9 Genome Editing Of Human Cullin‐3 In Vivo Enhances The Expression Of Bona Fide Cullin‐3 Substrates

Human patients with de novo loss‐of‐function mutations in Cullin‐3 (Cul3) resulting in deletion of exon 9, exhibit severe early onset hypertension mediated in part by impaired ubiquitination of WNK4. The objective of this study was to determine if genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR‐associated (Cas9) technique could permanently introduce deletion and insertion mutations into Cul3 using human embryonic kidney cell line (HEK293T). We hypothesize that genome editing of Cul3 would result in a loss‐of‐function phenotype leading to accumulation of Cul3 substrates. Using a plasmid vector expressing Cas9 enzyme and a GFP selectable marker, CRISPR guide RNA specifically targeting exon 5 and 7 of endogenous Cul3 were cloned into the vector and confirmed by sequencing. HEK293T cells were transfected and positive cells were sorted for GFP by flow cytometry. Using polymerase chain reaction (PCR), Cul3 exon 5 and 7 loci were amplified, cloned into a pCRII‐TOPO vector and sequenced. We found that genome editing induced both monoallelic and biallelic mutations in Cul3 upstream of the protospacer adjacent motif (PAM) in several HEK293T clones. This resulted in a loss of Cul3 protein expression. Interestingly, loss of Cul3 protein expression correlated with accumulation of RhoA, WNK4, and Cyclin E, all bona fide Cul3 substrates. Further, immunocytochemistry staining using confocal microscopy demonstrated significantly enhanced RhoA expression in Cul3‐edited cells compared to wildtype. These studies show that genome editing of Cul3 results in loss‐of‐function phenotype. Future studies will focus on generating HEK293T cells lacking exon 9 of Cul3.