Xing De, Bai Tao, Neyisci Ozlem, Paylakhi Seyedeh Zahra, Duval Alexander J, Tekin Yasemin, Adli Mazhar
Robert Lurie Comprehensive Cancer Center, Department of Obstetrics and Gynecology, Feinberg School of Medicine at Northwestern University, Chicago, IL, USA.
Nat Commun. 2025 Jul 18;16(1):6631. doi: 10.1038/s41467-025-61848-1.
Biological mechanisms are inherently dynamic, requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales, making them unsuitable for studying dynamic processes or characterizing essential genes, where chronic depletion can cause cell death. We compare five inducible protein degradation systems-dTAG, HaloPROTAC, IKZF3, and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2-evaluating degradation efficiency, basal degradation, target recovery after ligand washout, and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However, AID 2.0's higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations, we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants, including S210A, that significantly enhance the overall degron efficiency. The resulting degron system, named AID 2.1, maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout, enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts.
生物机制本质上是动态的，需要精确且快速的操作才能进行有效的表征。传统的基因操作在较长的时间尺度上起作用，这使得它们不适用于研究动态过程或表征必需基因，因为长期的基因敲除可能导致细胞死亡。我们比较了五种诱导型蛋白质降解系统——dTAG、HaloPROTAC、IKZF3以及使用OsTIR1和AtFB2的两种生长素诱导降解子（AID）——评估降解效率、基础降解、配体洗脱后的靶点恢复情况以及配体的影响。该分析确定基于OsTIR1的AID 2.0是最强大的系统。然而，AID 2.0较高的降解效率伴随着靶点特异性基础降解和较慢的恢复速率。为了解决这些局限性问题，我们采用碱基编辑介导的诱变，随后进行几轮功能选择和筛选。这种定向蛋白质进化产生了几种功能获得性的OsTIR1变体，包括S210A，它们显著提高了整体降解子效率。由此产生的降解子系统，命名为AID 2.1，在基础降解最小的情况下维持有效的靶点蛋白敲除，并且在配体洗脱后恢复更快，从而能够对必需基因进行表征和拯救实验。我们的比较评估和定向进化方法为在动态生物学背景下研究基因功能提供了一个参考数据集和改进的降解子技术。
