Double CRISPR/Cas9-knockout of ER chaperones, ERGIC transporters and GABARAPs reveal additive and dominant effect on FVIII secretion

Introduction: We previously demonstrated differential effects of single gene knockouts (SKOs) on FVIII secretion. SKOs of ER, ERGIC and autophagy-related proteins showed increasing (Calnexin, GABARAPL1, GABARAPL2, ATG7) or decreasing (Calreticulin, LMAN1, MCFD2, GABARAP) FVIII secretions. To further investigate potential additive or dominant effects, we generated double knockout (DKO) combinations.

Method: We have used CRISPR/Cas9 based technology for knockout generation in HEK293 cells, two-stage chromogenic assays to measure FVIII activity and secretion, and immunofluorescence (IF) imaging to asses FVIII intracellular localization, according to the standardized protocols established in our laboratory. Statistical analysis and Data visualization were carried out using GraphPad PRISM, Word Excel, Zen Blue 2.6 pro (Pearson’s Correlation Coefficients: PCC), and Qlucore omics explorer 3.6 softwares.

Results: We report here the generation of eight DKO combinations, confirmed by sequencing, western blot, and immunofluorescence (IF) staining. The effect of these DKOs on FVIII secretion divided them into three categories. The first category, exhibiting ahomogeneous effect, includes CANX/ATG7^-/-, CANX/GABARAPL1^-/-, and CALR/GABARAP^-/-, where FVIII activity values of both SKOs and DKOs were similar. The second category, showing an additive effect, includes LMAN1/MCFD2^-/- and CANX/GABARAP^-/-, where the DKOs’ effects on FVIII secretion were intermediate between the two SKOs. The third category consists of LMAN1/CANX^-/-, CALR/CANX^-/-, and GABARAP/GABARAPL1^-/-, where the DKOs’ effects on FVIII activity were strongly skewed toward one of the SKOs, which we refer to as the dominant effect ([Fig. 1]).

Fig. 1  The effects of single and double knockouts on FVIII activity levels, measured as percentage increase or decrease using a two-stage chromogenic assay, and FVIII co-localization with Cis- and Trans-Golgi markers (GM130 and TGN46), analyzed by showing percentage Pearson’s Correlation Coefficients (PCC) changes using Zen Blue 2.6 Pro and Excel software.

Conclusion: These findings highlight both additive and dominant influence in the generated DKOs, suggesting potential in-vivo effects of tissue-specific expression combinations of such proteins on FVIII secretion.