A Robust Protocol for CRISPR-Cas9 Gene Editing in Human Suspension Cell Lines

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems enable precise gene editing in mammalian cells.
• Existing CRISPR-Cas9 protocols primarily focus on adherent cell lines, leaving a gap for suspension cell applications.
• Suspension cell lines, common in hematolymphoid research, present unique challenges for gene editing experiments.

Purpose of the Study:

• To describe a comprehensive protocol for targeted gene manipulation using CRISPR-Cas9 in suspension cells.
• To provide optimization strategies for adapting the protocol to diverse suspension cell lines.
• To facilitate gene editing in suspension cells for researchers new to or optimizing CRISPR-Cas9 workflows.

Main Methods:

• Detailed protocol for transient expression of Cas9 nuclease and guide RNAs via electroporation (Neon™ or Nepagene).
• Workflows for generating single-cell clones using Fluorescence-Activated Cell Sorting (FACS) or manual dilution.
• Screening methods for identifying homozygous knockout clones, including PCR and Sanger sequencing.

Main Results:

• Successful implementation of CRISPR-Cas9 gene editing in B cell lymphoma suspension cells.
• Demonstrated workflows for single-cell cloning and confirmation of gene knockouts.
• Provided optimization insights for broader applicability across different suspension cell types.

Conclusions:

• The developed protocol effectively enables targeted gene editing in suspension cells.
• This resource aids researchers in establishing and optimizing CRISPR-Cas9 gene editing in challenging suspension cell models.
• The protocol promotes wider adoption of CRISPR-Cas9 technology in hematolymphoid and other suspension cell research.