Massively parallel screening of base editors in human pluripotent stem cells via virus-like particles

With the potential to differentiate into any cell type in the body and the ability for infinite self-renewal, human pluripotent stem cells (hPSCs) hold great promise for applications in cell replacement therapy, tissue engineering, and modeling human genetic diseases. The majority of the human disease mutations are single-nucleotide variants (SNVs), which have led the genome editing field to develop CRISPR-Cas9 base editors to enable SNV correction. However, the efficacy and precision of CRISPR base editors remain to be improved, especially in hPSCs. To address this, we have established a platform for screening base editor variants to identify the top performers in hPSCs using a virus-like particle (VLP) system. VLPs are self-assembling protein structures that mimic viral architecture while encapsulating cargo molecules produced by host cells. Although they retain the ability to transduce mammalian cells, VLPs are non-infectious and incapable of replication, as they lack viral genetic material. Compared to conventional delivery systems, VLPs offer several advantages: they can accommodate larger cargo sizes, efficiently package ribonucleoproteins (RNPs) to reduce off-target effects and can be pseudotyped with specific glycoproteins to direct cargo selectively to target cells. In parallel, we examined how modulation of DNA damage response and base excision repair pathways, including p53 inhibition and UGI, influences editing outcomes in hPSCs within a transient delivery framework. Together, this thesis establishes a scalable, massively parallel platform for interrogating genome-editing outcomes in human pluripotent stem cells, providing a foundation for addressing key bottlenecks in editing efficiency, delivery, and cell survival.