Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for the immune response to cancer and pathogen infection. Here, we discover that cGAS-DNA phase separation is required to resist negative regulation and allow efficient sensing of immunostimulatory DNA. We map the molecular determinants of cGAS condensate formation and demonstrate that phase separation functions to limit activity of the cytosolic exonuclease TREX1. Mechanistically, phase separation forms a selective environment that suppresses TREX1 catalytic function and restricts DNA degradation to an outer shell at the droplet periphery. We identify a TREX1 mutation associated with the severe autoimmune disease Aicardi-Goutières syndrome that increases penetration of TREX1 into the repressive droplet interior and specifically impairs degradation of phase-separated DNA. Our results define a critical function of cGAS-DNA phase separation and reveal a molecular mechanism that balances cytosolic DNA degradation and innate immune activation.

细胞溶质 DNA 的环状 GMP-AMP 合酶 (cGAS) 识别对于对癌症和病原体感染的免疫反应至关重要。在这里，我们发现需要 cGAS-DNA 相分离来抵抗负调节并允许有效地检测免疫刺激 DNA。我们绘制了 cGAS 凝聚物形成的分子决定因素，并证明相分离功能可以限制胞质外切核酸酶 TREX1 的活性。从机制上讲，相分离形成了一种选择性环境，抑制了 TREX1 的催化功能，并将 DNA 降解限制在液滴外围的外壳中。我们确定了一个TREX1与严重的自身免疫性疾病 Aicardi-Goutières 综合征相关的突变，增加了 TREX1 对抑制性液滴内部的渗透，并特别损害了相分离 DNA 的降解。我们的结果定义了 cGAS-DNA 相分离的关键功能，并揭示了平衡细胞溶质 DNA 降解和先天免疫激活的分子机制。