Osteoarthritis (OA) is the most common joint disorder worldwide, and pregnancy-associated plasma protein A (PAPPA), a metalloproteinase that increases insulin-like growth factor 1 (IGF-1) bioavailability, has been identified as an important OA risk gene. Knocking out PAPPA in primary human articular chondrocytes (pHACs) and evaluating the changes in these cells within an in vitro monolayer is a means to understand the direct, causal role of PAPPA in OA, but obtaining high-effect-size, physiologically representative readouts from this system remains challenging. Residual functional PAPPA in the treated population, dedifferentiation of chondrocytes in monolayer culture, and instability of chondrocyte micromasses are major barriers. This study aimed to validate the increase in CRISPR editing efficiency by the dual guide design and to improve the stability of chondrocyte micromass culture. Detailed manual deconvolution of alleles in edited single-cell-derived colonies and the use of the Inference of CRISPR Edits (ICE) tool in the bulk population allow comparison between single- and dual-guide editing. To improve micromass stability, a custom poly(2-hydroxyethyl methacrylate) (pHEMA) coating protocol was developed using a laser-cut mold that confines the coating to the brim of the well and limits cell migration. Dual-guide targeting achieved complete editing across all 24 deconvoluted alleles, a marked improvement over single-guide efficiency, which ranged from 60%~80% KO, although mRNA- and protein-level assays did not yet corroborate this advantage. The custom coating method substantially improved the uniformity and resolution of the micromasses, validated by a significant increase in the difference in Alcian Blue staining signal between the micromass center and periphery. Together, these optimizations advance the workflow for CRISPR-based functional study of PAPPA in pHACs and offer better versatility in the choice of functional experiments.
Alex Li (2026) studied this question.