Induced Pluripotent Stem Cells Meet Genome Editing

Abstract

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.

Figure 1. Overview of the iPSC technology

Patient cells can be reprogrammed into iPSCs using optimized reprogramming protocols that involve small molecules, microRNAs, and combinations of reprogramming factors. iPSCs can be differentiated into somatic cells that could be used either in transplantation therapies or alternatively to model human diseases.

Figure 2. Genome editing applications in hiPSCs

Genome editing allows for the genetic modification of hiPSCs. The top panel (left side) depicts examples of reverse genetic approaches to study hPSCs using genome editing. Gene expression can be modulated (activated or repressed: CRISPRi and CRISPRa) by reversibly targeting their endogenous promoter. Genes can be inserted to generate reporter genes or to achieve ectopic expression. Genetic information can be deleted or inverted and modifications as small as single base pairs changes can be generated to introduce mutations, polymorphisms or repair disease relevant mutations. The resulting genetically engineered hPSCs differ from wild type cells exclusively at the edited locus and are otherwise isogenic (bottom left). Parallel differentiation of these isogenic cell lines into disease relevant cell-types can provide the basis for the phenotypic analysis of disease specific cellular pathologies. Phenotypes found in these cells can be directly attributed to the genetic manipulation. In addition, forward genetic approaches to study hPSCs (top right panel) became available with the development of genome editing as a screening tool. Bulk transduction of hPSCs with either Cas9 or dCas9 in combination with genome wide barcoded sg RNA libraries --“CRISPR cutting, CRISPRi and CRISPRa”—can be used to identify genes who’s loss- or gain-of-function changes the cellular representation within the infect cell pool. Enrichment or depletion of sgRNAs can be determined by sequencing of the sgRNAs, yielding candidate genes of interest (bottom right panel).

Figure 3

Strategy to generate isogenic iPSCs that differ at multiple risk alleles. GWAS have identified genomic loci that may slightly increase the risk of developing a sporadic disease. The key challenge of using patient-derived iPSCs to get mechanistic insight into risk alleles is to create meaningful control cells. CRISPR/Cas9 mediated gene editing would allow to exchange risk (red squares) and protective (green squares) alleles and to generate appropriate control cells that differ exclusively at the risk loci under study.