CAREER: Develop PROTAC-CID, a Novel Small Molecule Inducible Platform for Controllable Gene Expression in Mammalian Cells

在哺乳动物细胞中很难控制基因的表达。实现这一点是开发有效的细胞疗法的关键。小分子可以施加这种控制。不幸的是，小分子往往是有毒的，往往具有有限的开关能力。该项目的目的是开发一种用于哺乳动物细胞的改进的小分子系统。该系统将是可调的，而不是开/关。高度敏感，它将与多个基因相互作用，指导更复杂的行为。来自弱势群体的本科生和高中教师将学习合成生物学和CRISPR技术。针对嵌合体的蛋白分解(PROTAC)-化学诱导二聚化(CID)系统将被开发为敏感、多重、可调节和安全的工具包，以控制哺乳动物细胞中的基因表达。将PROTAC-CID与基因组编辑相结合将使精确的基因组修改成为可能。这将大大提高在基础和治疗应用中使用基因组编辑的准确性和安全性。具体的研究目标是：1)建立一种新型的PROTAC-CID诱导基因表达系统；2)设计多种可诱导基因调控；3)设计高诱导和低基础水平的CRE重组酶表达PROTAC-CID系统；4)开发PROTAC-CID可诱导CRISPR碱基编辑。成功的完成将导致新的PROTAC-CID可诱导的基因表达平台，用于在活的真核细胞中进行多基因调控。这些系统将为实现高诱导和低基础水平的基因表达的方法提供深入的见解。最后，将PROTAC-CID系统应用于CRISPR基础编辑，将允许在定义的时间进行可诱导的基因组DNA修改，以减少偏离目标的影响，并提高基因组编辑的准确性和安全性。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

It is difficult to control gene expression in mammalian cells. Accomplishing this is key to developing effective cell therapies. Small molecules can exert that control. Unfortunately, small molecules are often toxic, and tend to have a limited, on-off capability. The aim of this project is to develop an improved small molecule system for mammalian cells. This system will be tunable instead of on/off. Highly sensitive, it will interact with multiple genes to direct more complex behaviors. Undergraduates from underrepresented groups and high school teachers will learn techniques of synthetic biology and CRISPR technology.Proteolysis Targeting Chimeric (PROTAC)-Chemically Induced Dimerization (CID) systems will be developed as sensitive, multiplex, tunable, and safe toolkits to control gene expression in mammalian cells. Combining PROTAC-CID with genome editors will enable precise genomic modification. This will greatly increase the accuracy and safety of using genome editors for basic and therapeutic applications. The specific research goals are to 1) establish a novel PROTAC-CID inducible gene expression system; 2) engineer multiplex inducible gene regulations; 3) design high-induction and low-basal level PROTAC-CID systems for Cre recombinase expression, and 4) develop PROTAC-CID inducible CRISPR base editors. Successful completion should result in novel PROTAC-CID inducible gene expression platforms for multiplex gene regulations in living eukaryotic cells. These systems will provide deep insights into the approaches to achieve high-induction and low basal-level gene expressions. Finally, applying PROTAC-CID systems to CRISPR base editors will permit inducible genomic DNA modification at a defined time to reduce the off-target effect and increase the accuracy and safety in genome editing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.