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A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Published on: July 12, 2017
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A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.
Alessandra Merenda1, Amanda Andersson-Rolf1, Roxana C Mustata2
1Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge; Department of Genetics, University of Cambridge.
Journal of Visualized Experiments : Jove
|July 27, 2017
Summary
This study introduces a new CRISPR method for simultaneously knocking out multiple genes in mouse organoids. This technique enhances gene function studies by overcoming limitations of single gene knockouts, enabling faster discovery.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Loss-of-function studies are crucial for understanding gene function.
- Paralogous genes in higher eukaryotes can mask phenotypes in single gene knockout studies.
- CRISPR/Cas9 technology has advanced gene editing for functional genomics.
Purpose of the Study:
- To develop a rapid cloning method for guide RNA (gRNA) concatemers for multi-gene knockouts.
- To enable simultaneous knockout of up to four genes in a single transfection.
- To improve the efficiency of genetic studies in higher eukaryotes.
Main Methods:
- Developed a novel Golden Gate shuffling reaction for gRNA concatemerization.
- Utilized a pre-designed retroviral vector for cloning multiple gRNAs.
- Optimized electroporation for efficient transfection in mouse small intestinal organoids.
Main Results:
- Successfully created a method for concatenating up to four gRNAs into a single vector.
- Demonstrated simultaneous knockout of gene pairs (Axin1/2 and Rnf43/Znrf3) in mouse intestinal organoids.
- Achieved high efficiency in multi-gene knockouts and electroporation.
Conclusions:
- The developed method allows rapid, simultaneous multi-gene knockouts in organoids.
- This approach overcomes limitations posed by gene compensation in functional studies.
- Facilitates deeper understanding of gene function and biological pathways, such as Wnt signaling.
