Background: The Fluorescent Ubiquitination-based Cell-Cycle Indicator (FUCCI) facilitates live monitoring of the cell cycle stages. The FUCCI reporter is founded on the fluctuations of human Geminin and Cdt1 protein levels during the cell cycle, mediated by the activity of E3 ligases APC and CUL4. These substrates are associated with fluorescent reporters AzaleaB5 and h2-3, allowing for the visual monitoring of the cell cycle stages via microscopy and enabling more straightforward investigation of cell cycle properties in various developmental stages and cell types.

Objective: This study aims to establish a collection of knock-in human induced pluripotent stem cell lines carrying the FUCCI-CA reporter using the SpCas9 nuclease.

Methods: The FUCCI-CA cassette was inserted into the AAVS1 safe-harbour locus using CRISPR-Cas9. Integration of the construct was evaluated through microscopy, PCR and Sanger sequencing. The pluripotency of the obtained cell lines was confirmed through flow cytometry, immunocytochemistry, and differentiation into three germ layers.

Results: Genome editing did not negatively affect the pluripotency of the obtained cell lines as assessed by flow cytometry and immunocytochemistry. The cells maintained their ability to differentiate into all three germ layers. Successful integration of the FUCCI-CA cassette was confirmed with PCR, and the expression of the fluorescent reporters AzaleaB5 and h2-3 was verified by microscopy.

Conclusion: The generated FUCCI-CA-expressing cell lines could be utilised as a platform for studying the properties of the cell cycle of iPS cells.