Li, H.; Liu, H.; Xu, W.; Zeng, Y.; Huang, P.; Guo, J.; Cai, B.; Chen, Y.; Lin, Y.; Zhang, C.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of low-density lipoprotein (LDL) cholesterol metabolism, yet the functional consequences of many clinically observed PCSK9 variants remain unknown. To establish a rigorous system for quantitative variant assessment, we generated a PCSK9 knockout (KO) HepG2 cell line through CRISPR/Cas9-mediated deletion of exons 2-8, effectively removing both the prodomain and catalytic regions required for PCSK9 function. This null background enabled systematic functional mapping of wild-type (WT) PCSK9 and multiple clinically relevant variants representing well-characterized, recurrent, and previously understudied alleles. Functional assays revealed pronounced heterogeneity among variant activities. The classical gain-of-function (GOF) variants D374Y and R496W exhibited robust suppression of LDL uptake, whereas A443T--an infrequently reported and previously uncharacterized variant--demonstrated a loss-of-function (LOF)-like phenotype with significantly enhanced LDL uptake. Additional poorly characterized variants, including V4I, R104C/V114A, and R496W/N425S, displayed minimal functional profiles, providing novel mechanistic insights. Surface LDL receptor (LDLR) levels generally correlated with LDL uptake but revealed unique patterns for specific variants. This KO-based rescue system provides a high-resolution framework for mechanistic classification of both established and poorly characterized PCSK9 variants, bridging the gap between genetic discovery and functional interpretation while supporting precision lipid-lowering strategies.