A simple, rapid, and efficient method for generating multigene-knockout culture cells by the CRISPR/Cas9 system

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• CRISPR-Cas9 gene editing allows for precise genetic modifications.
• Generating cell lines with multiple gene knockouts is crucial for understanding complex biological pathways.
• Existing methods for multiplex gene editing can be time-consuming and inefficient.

Purpose of the Study:

• To evaluate the efficacy of a simplified co-transfection method for simultaneous multiple-gene knockout in human cells.
• To establish a rapid and efficient protocol for generating multiplex gene-knockout cell lines.

Main Methods:

• HeLa cells were co-transfected with a mixture of pX330-based targeting plasmids and a puromycin resistance plasmid.
• Puromycin-resistant cells were selected to obtain Cas9/single-guide RNA (sgRNA)-transduced polyclonal cell populations.
• Western blot analysis and deep sequencing were used to confirm gene knockout efficiency and characterize mutations.

Main Results:

• Co-transfection of up to seven targeting plasmids effectively reduced the protein expression of targeted genes (p38α, p38β, JNK1, JNK2, Mnk1, ERK1, and mLST8).
• Analysis of 25 clones revealed knockout efficiencies ranging from 68% to 100% for the seven targeted genes.
• Six clones (24%) exhibited disruption of all targeted genes, with Cas9/sgRNA-induced mutations primarily consisting of small deletions or insertions.

Conclusions:

• Simple co-transfection enables efficient simultaneous multiple-gene knockout in human cell lines.
• This method provides a rapid, easy, and effective approach for generating multiplex gene-knockout cell lines.
• The findings facilitate the study of complex gene functions and pathways through combinatorial gene disruption.