Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.

Area of Science:

• Stem cell biology
• Genome editing
• Biotechnology

Background:

• CRISPR/Cas9 enables fluorescent protein knock-in into human pluripotent stem cells (hPSCs) for disease modeling and drug screening.
• Evaluating fluorescent reporter hPSC lines is crucial for high-content screening (HCS) applications.

Purpose of the Study:

• To assess the suitability of EGFP knock-in at the OCT4 and AAVS1 loci in hPSCs for HCS.
• To determine the stability and consistency of reporter gene expression for long-term screening platforms.

Main Methods:

• CRISPR/Cas9 mediated EGFP knock-in into the OCT4 and AAVS1 loci of hPSCs.
• Assessment of EGFP expression, pluripotency markers, and differentiation potential.
• Evaluation of reporter lines in multi-well formats for HCS.

Main Results:

• hPSC-OCT4-EGFP lines showed EGFP expression linked to pluripotency but lost expression during long-term culture.
• hPSC-AAVS1-EGFP lines exhibited stable and consistent EGFP expression throughout in vitro differentiation.
• The AAVS1 locus proved to be a safe and effective site for stable reporter gene integration without compromising hPSC quality.

Conclusions:

• The AAVS1 locus is a superior genomic site for generating stable reporter hPSC lines for long-term HCS applications using CRISPR/Cas9.
• Careful selection of reporter cell lines and genomic loci is essential for successful HCS and drug discovery.
• CRISPR/Cas9-mediated knock-in at the AAVS1 locus offers a robust platform for high-throughput screening and disease modeling.