CRISPR Manipulations in Stem Cell Lines.

Abstract

Insights into genome engineering in cells have allowed researchers to cultivate and modify cells as organoids that display structural and phenotypic features of human diseases or normal health status. The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. Modified organoids derived from patients' tissue cells are used as models to study diseases and test novel drugs. CRISPR-Cas9 technology has contributed to an explosion of advances that have the ability to edit genomes for the study of monogenic diseases and cancers. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of utmost importance as these cells carry the potential to be differentiated into any cell lineage. We describe recent developments that are broadening our understanding and extend DNA specificity, product selectivity, and fundamental capabilities. Furthermore, fundamental capabilities and remarkable advancements in basic research, biotechnology, and therapeutics development in cell engineering are detailed within this chapter. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter includes detailed protocols for the preparation of reagents to target loci of interest and transfection to genotype single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of ribonucleoprotein (RNP) delivered directly to cells.