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Marker-free coselection for CRISPR-driven genome editing in human cells.
Daniel Agudelo1, Alexis Duringer1, Lusiné Bozoyan1,2
1Centre Hospitalier Universitaire de Québec Research Center-Université Laval, Quebec City, Quebec, Canada.
Nature Methods
|April 19, 2017
Summary
This study introduces a new CRISPR-Cas9 method to efficiently enrich engineered cells for genome editing. This technique improves the recovery of cells modified via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) for research and therapies.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Targeted genome editing is crucial for creating cellular models and developing human cell-based therapies.
- Enhancing the efficiency of genome editing in cell populations is highly desirable for broader applications.
Purpose of the Study:
- To develop a versatile and robust coselection strategy for enriching cells undergoing genome editing.
- To increase the recovery rate of engineered cells using CRISPR-Cas9 and Cpf1 systems.
Main Methods:
- Designed a coselection strategy leveraging CRISPR-Cas9 and Cpf1 multiplexing capabilities.
- Utilized ouabain resistance conferred by modified Na+/K+ ATPase alleles to select for genetic modifications at unlinked loci.
- Applied the method to transformed cells, primary cells, and hematopoietic stem and progenitor cells.
Main Results:
- Successfully enriched cells with either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events.
- Demonstrated adaptability to various cell types, including primary human cells.
- Streamlined the incorporation of marker-free genetic modifications in human cells.
Conclusions:
- The developed coselection strategy significantly enhances the efficiency of genome editing.
- This method offers a broadly applicable and versatile tool for biological research and potential therapeutic applications.
- Facilitates marker-free genetic engineering in human cells, advancing cell-based therapies.