Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids

Area of Science:

• Genetics
• Molecular Biology
• Stem Cell Biology

Background:

• Genetic modification, including gain- and loss-of-function studies, is crucial for understanding gene function.
• Paralogous genes can cause genetic compensation, requiring knockout of all paralogs for clear phenotypic observation.
• CRISPR technology offers efficient gene editing but simultaneous multi-gene knockout in organoids was previously undescribed.

Purpose of the Study:

• To develop a rapid, scalable, and cost-effective method for generating multiple gene knockouts in organoids.
• To enable simultaneous targeting of multiple genes using CRISPR technology in organoid models.
• To demonstrate the utility of this method for biological pathway manipulation.

Main Methods:

• Developed a 'gRNA concatemer vector' by concatemerizing multiple gRNA expression cassettes.
• Utilized a single-step assembly of annealed synthetic DNA oligos into the vector.
• Applied the method for simultaneous knockout of up to four genes in organoids.

Main Results:

• Successfully generated a novel method for rapid, scalable, and cost-effective double gene knockouts in organoids.
• Demonstrated the ability to deliver multiple gRNAs simultaneously for efficient gene knockout.
• Achieved knockout of negative Wnt pathway regulators in small intestinal organoids, eliminating R-spondin1 dependence.

Conclusions:

• The developed gRNA concatemer vector method facilitates efficient simultaneous multi-gene knockout in organoids.
• This approach accelerates genetic studies in organoid systems.
• The method has significant implications for investigating gene function and biological pathways in complex cellular models.