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Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM24385 cells by flow cytometry and Sanger sequencing
Sarah L Inwood1, Linhua Tian1, Kirsten Parratt1
1Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Biotechniques
|June 15, 2022
Summary
This study presents a flow cytometry protocol to assess CRISPR/Cas9 gene editing in B-lymphoblast cells. This method supports the development of safe and effective CRISPR/Cas9-engineered therapies.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- CRISPR/Cas9 is a widely used genome editing tool for therapeutic applications.
- Limited long-term data exists on the genomic and phenotypic stability of CRISPR/Cas9 edited cells.
- Flow cytometry offers a potential method for characterizing edited cells in longitudinal studies.
Purpose of the Study:
- To describe a flow cytometry protocol for measuring CRISPR/Cas9 edits.
- To characterize edited cells using a well-established B-lymphoblast cell line (GM24385).
- To support the development of safe and effective CRISPR/Cas9-engineered therapies.
Main Methods:
- Utilized CRISPR/Cas9 genome editing technology.
- Employed flow cytometry for cell characterization.
- Used the GM24385 B-lymphoblast cell line.
Main Results:
- A detailed protocol for flow cytometry analysis of CRISPR/Cas9 edits was established.
- The protocol allows for the characterization of edited cells.
- Data supports the feasibility of using flow cytometry for longitudinal studies of edited cells.
Conclusions:
- Flow cytometry is a viable method for assessing CRISPR/Cas9 gene editing.
- This protocol can aid in evaluating the stability of edited cells.
- The findings contribute to advancing the safety and efficacy of CRISPR/Cas9-based therapies.
