CRISPR Screen Pinpoints Clusterin as Master Driver of Immune Aging in Blood Stem Cells

Aging blood stem cells (HSCs) are a root cause of immune decline in older adults, shifting production away from infection-fighting lymphocytes toward inflammation-promoting myeloid cells. This myeloid bias contributes to increased susceptibility to infection, hematologic malignancies like clonal hematopoiesis, and systemic functional decline. Despite the clinical importance of this phenomenon, the molecular drivers have remained largely unknown, limiting therapeutic strategies. This Harvard-led study set out to identify causal regulators using an unbiased in vivo CRISPR approach.

The team first compared transcriptomes of HSCs from young mice (2 months) and old mice (24 months), identifying 77 significantly upregulated genes in aged HSCs (FPKM > 1, adjusted P < 0.05, log2FC > 0.5). Cross-referencing with a published 740-gene aging signature narrowed the list to 50 candidates, and a scoring system based on expression level, fold change, adjusted P-value, and consistency yielded 23 top candidates for functional screening. Using a barcode-based in vivo CRISPR loss-of-function screen — three sgRNAs per gene, five barcodes each — they introduced gene knockouts into aged Cas9-expressing HSCs, transplanted them into lethally irradiated recipients, and analyzed hematopoietic lineage outputs after 5 months. Four pairwise comparisons (mature cells vs. start-point HSCs, myeloid vs. lymphoid, and endpoint HSCs vs. mature lineages) converged on clusterin (Clu) and Cd38 as the top hits whose loss increased lymphoid differentiation.

Loss-of-function validation confirmed that Clu knockout in aged HSCs reduced myeloid output in vitro and in vivo, while Clu overexpression in young HSCs recapitulated the aged, myeloid-biased phenotype. Mechanistically, the team found that Clu physically interacts with Mitofusin-2 (Mfn2), a key mediator of mitochondrial outer membrane fusion. Clu upregulation in aged HSCs promotes mitochondrial hyperfusion — abnormally elongated mitochondrial networks detectable by electron microscopy — which drives excessive oxidative phosphorylation (OXPHOS) and elevates reactive oxygen species (ROS). This OXPHOS-driven ROS surge activates p38 MAPK, which in turn upregulates C/EBPβ (Cebpb), a master transcription factor for myeloid differentiation. Clu knockout in aged HSCs attenuated OXPHOS, improved mitophagy (clearance of damaged mitochondria), reduced p38 activation, and lowered Cebpb expression — all consistent with a reversal of the aged phenotype.

In transplantation experiments, Clu-depleted aged HSCs transferred into middle-aged recipient mice (12 months) produced balanced hematopoiesis with restored lymphoid output and improved physical performance metrics in recipients. This is particularly significant because it demonstrates functional rejuvenation of the systemic immune and hematopoietic milieu, not merely a molecular correction. The gain-of-function experiments with Clu overexpression in young HSCs, which phenocopied aged myeloid bias, further strengthened the causal relationship and ruled out confounding age-related variables.

The study has important implications for understanding and potentially treating age-related immune dysfunction. The Mfn2-OXPHOS-p38-Cebpb axis identified here represents a linear, druggable pathway connecting a secreted chaperone protein to a transcriptional myeloid commitment program. Existing pharmacological tools targeting mitochondrial dynamics (Mfn2), OXPHOS, p38 MAPK, or C/EBPβ could in principle be tested as rejuvenating interventions. Clusterin itself is already recognized as a biomarker elevated in Alzheimer's disease and other aging-related conditions, suggesting this pathway may have broad relevance across aging biology. While the work is conducted in mice and will require human validation, the clear genetic causality established here offers a strong translational foundation.