DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.

Area of Science:

• Stem cell biology
• Genomic engineering
• Molecular biology

Background:

• Human pluripotent stem cells (hPSCs) require advanced genomic engineering tools.
• Efficient and site-specific gene modification is crucial for unlocking hPSC potential.

Purpose of the Study:

• To develop a novel system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
• To establish a safe and reliable genomic locus for robust gene expression in hPSCs.

Main Methods:

• Identification of the H11 locus on chromosome 22 as a safe, transcriptionally active site for gene insertion.
• Establishment of recipient cell lines with a 'landing pad' cassette at the H11 locus using homologous recombination.
• Application of the Dual Integrase Cassette Exchange (DICE) system utilizing phiC31 and Bxb1 integrases for site-specific gene insertion.

Main Results:

• The DICE system achieved 100% specificity for gene insertion at the H11 locus.
• Genes, including mCherry and neural transcription factors, were successfully inserted into ESCs and iPSCs.
• The system demonstrated control over gene content, direction, and copy number.

Conclusions:

• The DICE system provides rapid, efficient, and precise gene insertion in ESCs and iPSCs.
• This technology is well-suited for repeated genetic modifications at the same locus.
• The H11 locus serves as an effective site for ubiquitous gene expression in hPSCs.