Epigenetic CRISPR Screening of 9p21.3 Non-Coding Regions identifies Cis-Regulatory Elements of P16INK4a and P15INK4b Controlling Cellular Senescence

Cellular senescence is a hallmark of aging and a promising target for extending human healthspan. Senescence is often accompanied by upregulation of the key senescence marker gene CDKN2A, yet the mechanism underlying its transcriptional activation remains unclear due to complex cis-regulations within the 9p21.3 locus. Here, we performed complementary CRISPR activation and interference screens in human mesenchymal stromal cells (MSCs) to systematically map non-coding cis-regulatory elements (CREs) at this locus that epigenetically regulate senescence. This approach revealed senescence-regulating CREs (SenReg-CREs) that bidirectionally modulate senescence through P16INK4a and P15INK4b. Notably, we identified a primate-specific short interspersed nuclear element (SINE) MIR3 embedded within the most potent distal SenReg-CRE. Deletion of this SINE:MIR3 accelerated senescence, revealing its potential insulator function in restraining CDKN2A/CDKN2B activation. Collectively, these findings reveal novel mechanisms underlying senescence-associated transcriptional activation of CDKN2A/CDKN2B and demonstrate that senescence is malleable through manipulation of regulatory element activity, highlighting the potential of epigenetically targeting these SenReg-CREs for senomorphic interventions.