Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools

Abstract

Summary: Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation.For complete details on the use and execution of this protocol, please refer to Geurts et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cancer
• Genetics
• CRISPR
• Stem Cells