Jan 10, 2026

Public workspaceGeneration of Knockout Cell Lines Using CRISPR–Cas9 Technology

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlky5m5g5r/v1
  • Ali Ghoochani1,2,3,4,
  • Monther Abu-Remaileh1,2,3,4
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlky5m5g5r/v1
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Generation of Knockout Cell Lines Using CRISPR–Cas9 Technology. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlky5m5g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 08, 2025
Last Modified: January 10, 2026
Protocol Integer ID: 222025
Keywords: cas9 genome editing, generation of gene knockout cell line, gene knockout cell line, using crispr, generation of knockout cell line, crispr, cas9 technology, cas9 technology this protocol, knockout cell line, sgrna design, cas9, genome, gene, cell
Abstract
This protocol describes the generation of gene knockout cell lines using CRISPR–Cas9 genome editing. The procedure involves sgRNA design, cloning into a Cas9-GFP expression vector pX458, transfection, single-cell sorting, colony expansion, and validation by Sanger sequencing and ICE analysis.
Materials

·       pX458 vector (expressing Cas9 and GFP) pSpCas9(BB)-2A-GFP (PX458) (Addgene #48138)
·       Targeting sgRNA oligos
·       XtremeGene 9 transfection reagent
·       DMEM with 30% FBS
·       Penicillin/Streptomycin
·       96-well plates
·       Competent cells (recombination-deficient)
·       T4 DNA ligase buffer (10X)
·       BbsI enzyme
·       PCR reagents and primers
·       Sanger sequencing services
·       ICE indel analysis tool (https://synthego.com)
Troubleshooting
Design sgRNAs




Annealing sgRNAs

• 1 µl Oligo 1 (100 μM) • 1 µl Oligo 2 (100 μM) • 1 µl 10X T4 Ligation Buffer (NEB) • 7 µl ddH₂O • Total: 10 µl


Thermocycler conditions for annealing:
• 37°C for 30 min • 95°C for 5 min • Ramp down to 25°C at 5°C/min
Golden Gate Ligation Reaction
• 1 µl of 1:100 diluted annealed oligos • X µl backbone vector (20 ng) • Add water to 16 µl total • 2 µl 10× T4 ligase buffer • 1 µl BbsI
Thermocycler conditions for ligation:
• 42°C for 5 min • 42°C for 5 min • 16°C for 5 min • 55°C for 10 min
For 30 cycles
Transformation and Maxiprep
Transform 2 µL of the ligation reaction into 50 µL competent cells. Also include a negative control ligation. Continue transformation.
Purify the ligated sgRNA-pX458 construct using a Maxiprep kit
Transfection and Single-Cell Cloning
Transfect cells with the pX458-sgRNA plasmid using XtremeGene 9 at a 1:3 DNA-to-reagent ratio.   
 After 48 hours, sort GFP-positive cells using a flow cytometer into 96-well plates (1 cell/well) containing 200 µL of DMEM with 30% FBS and penicillin/streptomycin.
Incubate at 37°C and 5% CO₂ to allow colony formation
Once colonies are visible, harvest cells for genomic DNA extraction
Validation of Knockout
 PCR amplify the sgRNA target region from each clone.
   
Submit PCR products for Sanger sequencing.
Analyze sequences using the Synthego ICE indel deconvolution tool.