Dear Editor, Atherosclerotic plaques are formed by lipid-rich molecules on the arterial wall that narrow the arteries over time, leading to myocardial infarction and stroke. Unraveling the origin and plasticity of various cell types that critically participate in all phases of plaque formation and destabilization is essential in understanding the pathogenesis of atherosclerosis. Smooth muscle cells (SMCs) in the plaques are derived from preexisting SMCs in the medial layer of vessel, and a subset of these cells in the advanced atherosclerotic plaques can differentiate into macrophage-like cells or foam cells 1–3. Genetic lineage tracing studies demonstrate that some descendants of SMCs may no longer express SMC markers and convert to macrophage-like cells 2, 3. However, whether these SMC-derived macrophage-like cells adopt a stable or transient macrophage cell fate during the progression of atherosclerosis remains controversial and unclear 4. Here, we developed a dual genetic lineage approach to specifically trace lineage conversion of SMC-derived macrophage-like cells, if any, and found that a subset of these cells re-adopted the SMC lineage in the fibrous cap, contributing to fibroblasts and pericytes in the plaque. To specifically trace SMC-derived macrophage-like cells, we used dual orthogonal recombination systems of Dre-rox and Cre-loxP as previously reported 5. We first generated a Myh11-Dre knock-in mouse line by homologous recombination using CRISPR/Cas9 and crossed it with the rox reporter R26-rox-ZsGreen line (Supplementary Fig. S1a, b). Immunostaining for ZsGreen and SMC markers such as SMA, SM22, CNN1, and smMHC revealed that Myh11-Dre efficiently and specifically targeted aortic SMCs (Supplementary Fig. S1c, d). We next generated a CD11b-CrexER knock-in mouse line by targeting Cre-rox-ER-rox (CrexER) into the CD11b gene locus (Supplementary Fig. S1e) and crossed it with the loxP reporter R26-loxP-tdTomato line (R26-tdT, Supplementary Fig. S1f). Without tamoxifen treatment, we did not detect any tdTomato+ cell in the blood, spleen, or bone marrow, indicating no leakiness of CD11b-CrexER; while tamoxifen treatment resulted in specific labeling of CD11b+ cells (Supplementary Fig. S1g, h), indicating specificity. Having successfully generated the Myh11-Dre and CD11b-CrexER mouse lines, we crossed them to develop a sequential intersectional genetic strategy in which Myh11-Dre-mediated Dre-rox recombination first removed ER from the CrexER cassette, thus switching CD11b-CrexER into the CD11b-Cre genotype in smMHC+ SMCs (Step 1, Fig. 1 a). CD11b-expressing macrophages originating from smMHC+ SMCs were, therefore, genetically labeled by the R26-tdT reporter, and their subsequent cell fate in the atherosclerotic plaques could be traced and analyzed using tdTomato (Step 2, Fig. 1 a). By crossing with LDLR−/−, we generated the Myh11-Dre;