RAP1 is a well-known telomere-binding protein,but its functions in human stem cells have remained unclear.Here we generated RAP1-deficient human embryonic stem cells (hESCs) by using CRISPR/Cas9 technique and obtained RAP1-deficient human mesenchymal stem cells (hMSCs) and neural stem cells (hNSCs) via directed differentiation.In both hMSCs and hNSCs,RAP1 not only negatively regulated telomere length but also acted as a transcriptional regulator of RELN by tuning the methylation status of its gene promoter.RAP1 deficiency enhanced self-renewal and delayed senescence in hMSCs,but not in hNSCs,suggesting complicated lineage-specific effects of RAP1 in adult stem cells.Altogether,these results demonstrate for the first time that RAP1 plays both telomeric and nontelomeric roles in regulating human stem cell homeostasis.