A ribonucleoprotein-based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering.

Area of Science:

• * Molecular Biology
• * Biotechnology
• * Genomics

Background:

• * CRISPR/Cas9 knockout (KO) technology is vital for Chinese hamster ovary (CHO) cell engineering.
• * Efficiently targeting multiple genes (multiplex KO) is essential for advancing CHO cell line development.
• * Current methods require optimization for simultaneous or sequential targeting of numerous genes.

Purpose of the Study:

• * To develop and optimize a multiplex gene KO protocol for CHO cells.
• * To enable efficient targeting and KO of up to 10 genes using sequential transfections.
• * To establish a streamlined method for generating multiple gene knockouts in CHO cells.

Main Methods:

• * Utilized Cas9 protein and synthetic guide RNAs (gRNAs) for gene targeting.
• * Employed sequential transfections for delivering gRNAs and Cas9.
• * Verified gene deletions via Sanger sequencing (DNA level) and mass spectrometry (protein level).

Main Results:

• * Identified effective gRNA sequences through screening and Sanger sequencing indel analysis.
• * Successfully generated single cell clones with targeted deletion of all 10 genes.
• * Isolated 6 clones with complete 10-gene deletion out of 704 screened clones, confirming the decaplex gene editing strategy.

Conclusions:

• * The described sequential transfection method enables efficient decaplex gene editing in CHO cells.
• * This approach significantly reduces the time and effort for generating multiple gene knockouts.
• * The findings support rapid advancement of CHO cell engineering for biotechnological applications.