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

This protocol provides a step-by-step approach for generating single-gene knockout in hard-to-transfect suspension immune cell lines like THP1, specifically demonstrated by knocking out the GSDMD gene. By employing CRISPR-Cas9 system delivered via lentivirus, this protocol enables precise gene disruption through targeted single-guide RNAs (sgRNAs). Key steps include designing specific sgRNAs, cloning them into a CRISPR vector, viral packaging, and transducing the target cells, followed by selection and validation. This optimized protocol is particularly useful for functional studies in immune cells, allowing researchers to reliably explore gene function in complex cellular pathways. Key features • CRISPR-Cas9-based knockout strategy tailored for hard-to-transfect THP1 cells using lentiviral delivery. • Lentiviral transduction ensures stable gene delivery with high efficiency compared to other traditional methods like transfection and electroporation. • Stepwise validation through colony PCR, sequencing, and western blotting to confirm successful knockout. • Scalable approach, applicable to various cell models for functional genomic studies. Graphical overview.