Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools
Martina Celotti, Lucca L. M. Derks, Johan H. van Es, Ruben van Boxtel, Hans Clevers, Maarten H. Geurts
Abstract
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 • Base- and prime editor guide RNA design for isogenic organoid line generation • Electroporation-based transfection of CRISPR tools into organoids • Experimental setup to select for CRISPR-engineered organoids • Clonal isogenic line generation and Sanger-based validation of genome editing Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.