Abstract A166: CRISPR-Cas9 genome editing in iPSCs for functional genetic screening

Abstract

Induced pluripotent stem cells (iPSCs) are widely used for disease modeling, drug discovery, and cell therapy development. However, iPSCs are difficult to engineer with an efficient CRISPR/Cas9 system for functional genetic screens. We used WTC11 human iPS cells to generate functionally validated, lentiviral-transduced Cas9 lines, characterized by high gene-editing activity and sustained potential to differentiate. Flow cytometry was used to check for the expression of pluripotency markers Oct3/4, TRA1-60, and SSEA-4 in the iPS-Cas9 cells, and their actual pluripotency was then confirmed by testing the ability to differentiate into the three germ layers: ectoderm, mesoderm, and endoderm. Furthermore, genome-wide transcriptome analysis was performed to confirm the expression of germ layer-specific markers in the differentiated cells. Sustained Cas9 activity was confirmed in the differentiated cells. This study provides proof-of-principle that patient-derived iPSCs can be used to enable CRISPR/Cas9 functional genetic screening technology in reconstituted patient-specific tissues or disease models.

Citation Format: Nadja Isachenko, Dongfang Hu, Alex Chenchik, Paul Diehl, Donato Tedesco. CRISPR-Cas9 genome editing in iPSCs for functional genetic screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A166.