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

Molecules. 2022 Apr 9;27(8):2434. doi: 10.3390/molecules27082434.


Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC-OCT4-EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC-AAVS1-EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC-AAVS1-EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.

Keywords: AAVS1 locus; CRISPR/Cas9; EGFP reporter lines; OCT4 locus; genome editing; human pluripotent stem cells; phenotypic screening.

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Cell Differentiation / genetics
  • Cell Line
  • Gene Editing / methods
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Humans
  • Pluripotent Stem Cells*


  • Green Fluorescent Proteins