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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

670
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Related Experiment Video

Updated: Oct 19, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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A ribonucleoprotein-based decaplex CRISPR/Cas9 knockout strategy for CHO host engineering.

Joseph Carver1, Marie Kern1, Peggy Ko1

  • 1Cell Culture and Bioprocess Operations, Genentech, Inc., South San Francisco, California, USA.

Biotechnology Progress
|September 19, 2021
PubMed
Summary

This study presents a new CRISPR/Cas9 method for efficiently knocking out up to 10 genes in Chinese hamster ovary (CHO) cells. The sequential transfection approach speeds up multiplex gene editing for cell engineering.

Keywords:
CHO cellsCRISPR/Cas9gRNAmultiplex knockoutribonucleoprotein

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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.