CRISPR/dCas9 DNA methylation editing is heritable during human haematopoiesis and can impair differentiation in vivo [methylation array]

Ageing is associated with an abnormal increase of DNA methylation in human gene promoters, including in the bone marrow stem cells. DNA methylation patterns are further perturbed in haematological malignancies such as acute myeloid leukaemia (AML) but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show evidence that p15 methylation affects haematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNA methylation spreading beyond the location of the gRNA. 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, we identified how the 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 cells. We also observe significant changes in the proportions of multipotent and committed progenitor cells. This may have clinical relevance since we found that p15 promoter methylation is present in the peripheral blood of patients with clonal haematopoiesis. 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 haematopoiesis in vivo. Targeted bisulfite-sequencing data from the p15, p14 and p16 promoter after epigenetic editing or targeted-seq data at TET2 after genetic editing in haematopoetic stem and progenitor cells and in vitro experiments (colony forming unit assay) or in vivo engraftment into mice. Also targeted bisulfite sequencing of the p15 and p14 promoters in patients with Clonal Hematopoiesis

衰老与人类基因启动子（包括骨髓干细胞中）的DNA甲基化（DNA methylation）异常升高相关。血液系统恶性肿瘤（haematological malignancies）如急性髓系白血病（acute myeloid leukaemia，AML）中DNA甲基化模式进一步紊乱，但此类表观遗传改变（epigenetic changes）的生理学意义尚不明确。本研究通过对人类造血干/祖细胞（human stem/progenitor cells，HSPCs）进行表观遗传编辑（epigenetic editing），证实p15甲基化可在体内影响造血功能。我们利用dCas9-3A3L对CDKN2B（p15）启动子与ARF（p14）进行编辑，观察到DNA甲基化扩散至向导RNA（guide RNA，gRNA）结合位点以外的区域。研究发现，尽管采用的是瞬时递送系统，DNA甲基化在体外髓系分化（myeloid differentiation）过程中仍可维持，且p15启动子的高甲基化会降低基因表达。在体内，经编辑的人类HSPCs可植入小鼠骨髓，靶向DNA甲基化可在HSPCs中长期维持。此外，我们阐明了此类表观遗传改变在髓系与淋巴系细胞谱系（lymphoid lineages）中的保守性与传代模式。尽管髓系（CD33+）与淋巴系（CD19+）细胞的比例未受影响，但植入p15高甲基化细胞的小鼠中，单核细胞（CD14+）群体减少，粒细胞（CD66b+）群体增多。我们还观察到多能祖细胞与定向祖细胞的比例发生显著变化。鉴于我们在克隆性造血（clonal haematopoiesis）患者的外周血中检测到p15启动子甲基化，该研究结果或具备临床相关性。本研究证实，DNA甲基化可在人类HSPCs中被靶向并维持，并证明了p15位点异常DNA甲基化的功能相关性。由此推测，其他与衰老相关的异常DNA甲基化或许也可在体内影响造血功能。本研究附带的数据包括：表观遗传编辑后p15、p14及p16启动子的靶向亚硫酸氢盐测序（targeted bisulfite-sequencing）数据；造血干/祖细胞中TET2位点经遗传编辑后的靶向测序（targeted-seq）数据；体外实验（colony forming unit assay，集落形成单位实验）数据；以及小鼠体内植入实验数据。此外还包含克隆性造血患者p15与p14启动子的靶向亚硫酸氢盐测序数据。