Protocol for Generation of Single-Gene Knockout in Hard-to-Transfect THP1 Cell Lines Using CRISPR/Cas9

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Immune cell lines, such as THP1, are crucial for studying cellular pathways but are often challenging to genetically modify.
• Efficient gene knockout methods are essential for understanding gene function in these complex systems.

Purpose of the Study:

• To establish a reliable protocol for generating single-gene knockouts in hard-to-transfect suspension immune cells.
• To demonstrate the efficacy of a CRISPR-Cas9 system delivered via lentivirus for precise gene disruption in THP1 cells.

Main Methods:

• Design and cloning of single-guide RNAs (sgRNAs) into a CRISPR vector.
• Lentivirus production and transduction of THP1 cells for gene delivery.
• Selection and stepwise validation of gene knockout using colony PCR, sequencing, and Western blotting.

Main Results:

• Successful generation of single-gene knockout (GSDMD) in THP1 cells.
• Lentiviral delivery demonstrated high efficiency for gene editing in suspension immune cells.
• Comprehensive validation confirmed the accuracy and reliability of the knockout protocol.

Conclusions:

• This optimized protocol provides a scalable and efficient method for CRISPR-Cas9 mediated gene knockout in challenging immune cell lines.
• The protocol facilitates functional genomic studies by enabling precise gene disruption and subsequent analysis in immune cells.