CRISPR/dCas9 DNA methylation editing is heritable during human hematopoiesis and shapes immune progeny [RNA-seq]

Aging is associated with an abnormal increase of DNA methylation in human gene promoters, including in bone marrow stem cells. DNA methylation patterns are further perturbed in hematological malignancies such as acute myeloid leukemia (AML) but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNA methylation spreading beyond the gRNA location. We find that despite a transient delivery system, DNA methylation is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNA methylation is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNA methylation can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNA methylation on the p15 locus. As such, other ageing associated aberrant DNA methylation may impact hematopoiesis in vivo. RNA-seq experiment monocytes and granulocytes harvested from engrafted mice. We sequenced 20 samples in total (5 controls and 5 p15 edited for both cell types; 1 control sample for granulocytes failed during low input Smart-seq2 preparation), therefore we have 19 samples in total. Monocytes and granulocytes harvested from mouse bone marrow were prepared for RNA-seq using a low input Smart-seq2 protocol (library preparation and sequencing by Novogene).