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Updated: Mar 8, 2026
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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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One-step generation of conditional and reversible gene knockouts
Amanda Andersson-Rolf1,2, Roxana C Mustata1, Alessandra Merenda1,2
1Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.
Nature Methods
|January 31, 2017
Summary
CRISPR-FLIP enables efficient conditional gene knockouts in diploid cells. This CRISPR-Cas9 based strategy allows for rapid, scalable biallelic gene inactivation in various cell types, advancing functional genomics research.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Cell Biology
Background:
- Loss-of-function studies are crucial for understanding gene function.
- CRISPR technology has democratized genome editing but struggles with conditional gene inactivation in diploid cells.
Purpose of the Study:
- To develop an efficient, rapid, and scalable method for biallelic conditional gene knockouts in diploid and aneuploid cells.
- To introduce CRISPR-FLIP, a novel strategy for precise gene inactivation.
Main Methods:
- Co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette.
- Application of the CRISPR-FLIP strategy in various cell types including pluripotent stem cells, 3D organoids, and cell lines.
Main Results:
- Demonstration of an efficient and scalable method for biallelic conditional gene knockouts.
- Successful implementation of CRISPR-FLIP across diverse cell types.
Conclusions:
- CRISPR-FLIP provides a powerful tool for conditional gene inactivation in diploid cells.
- This strategy enhances the study of gene function in complex cellular systems and organoids.
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