CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

Objective: To gain mechanistic insights into the role of (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages.

Approach And Results: We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage loss-of-function phenotypes. was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of () had barely detectable LAL enzymatic activity. Control and IPSDM were loaded with [H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [H]-cholesterol to apolipoprotein A-I was abolished in IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [H]-cholesterol-labeled AcLDL, [H]-cholesterol efflux was, however, not different between control and IPSDM. (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and IPSDM. In nonlipid loaded state, IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and IPSDM. did not impact lysosomal apolipoprotein-B degradation or expression of , , and CONCLUSIONS: IPSDM reveals macrophage-specific hallmarks of deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated genetic variation in human macrophages.