Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology

Area of Science:

• Molecular Biology
• Genetics
• Stem Cell Biology

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems are powerful tools for gene function investigation.
• Human trophoblast stem cells (hTSCs) are crucial for studying placental development and function.
• Efficient genome editing and transcriptional regulation in hTSCs are essential for advancing research in this area.

Purpose of the Study:

• To establish an optimized workflow for efficient genome editing in hTSCs.
• To develop a method for transcriptional activation in hTSCs.
• To utilize a lentivirus-dependent system for these genetic modifications.

Main Methods:

• Employed a lentivirus-dependent delivery system for CRISPR/Cas9 components.
• Implemented optimized protocols for genome editing (gene targeting) in hTSCs.
• Developed and applied methods for assessing transcriptional activation in modified hTSCs.
• Established techniques to evaluate gene-targeted mutations.

Main Results:

• Achieved efficient genome editing in human trophoblast stem cells.
• Successfully demonstrated transcriptional activation in hTSCs using the developed workflow.
• Validated methods for evaluating the efficiency and accuracy of gene editing and transcriptional changes.

Conclusions:

• The presented lentivirus-based workflow provides an efficient means for genome editing and transcriptional activation in hTSCs.
• This optimized method facilitates the study of gene function in the context of human placental development.
• The developed evaluation techniques ensure reliable assessment of genetic modifications in hTSCs.