Advanced synthetic biology tools for genetic modification of human stem cells and their applications

The precise and efficient genetic modification of human induced pluripotent stem cells (hiPSCs) continues to present significant challenges, as existing methods are often limited by low efficiency, lengthy timelines, or technical complexity. This thesis addresses these limitations through the development of the STRAIGHT-IN platform, a method enabling the rapid and highly efficient targeted integration of large DNA payloads into the hiPSC genome. The STRAIGHT-IN platform enables the seamless insertion of one or two sizable genetic constructs in as little as two weeks, achieving integration efficiencies approaching 100%. This approach has been demonstrated in various biomedical applications, including drug screening, disease modeling, and forward programming, showcasing its versatility and potential to address current limitations in hiPSC genetic engineering. Together, these contributions highlight the potential of the STRAIGHT-IN platform, setting a new standard for genetic engineering in hiPSCs and opening new avenues for research and clinical applications.