Engineering of human-induced pluripotent stem cells for precise disease modeling

Stem cell technologies and gene editing techniques are two of the most promising recent developments in biomedicine. The ability to reprogram common human cells into induced pluripotent stem cells (hiPSCs) and turn them into the cells of interest has already become a powerful research tool, thus providing a unique platform for disease studies. In combination with the use of designer nucleases approach to repair or to introduce disease-causing mutations, both are valuable in developing personalized disease models. This chapter provides an overview on designer nucleases-based gene editing in hiPSCs, describing the principles of CRISPR/Cas systems along with consecutive methodological steps such as nucleases selection, isolation, and genotyping of modified hiPSC clones with emphasis on the crucial role of isogenic cell lines in disease modeling. Moreover, the production of rare or complex genotypes in patient cell lines requires efficient and streamlined gene editing technologies. However, precise genome editing applications rely on infrequent homology-directed repair (HDR), with the abundant nonhomologous end joining (NHEJ) formed indels presenting a barrier to achieving high rates of precise sequence modification. The methods presented here are supported by theoretical framework to allow for the incorporation of inevitable improvements to achieve either higher rates of gene editing by promotion of HDR over NHEJ or application of different CRISPR/Cas platforms for robust and multiplex gene editing, toward decoding of neurodevelopmental as well as for modeling of late onset disorders by fast-forwarding the biological clock. Due to easy in theory but laborious and inefficient in practice, the precise and efficient genome editing in hiPSCs could be only achieved by the proper combination of the described methods in the process. This eventually would lead to generation of wide range of disease models for decoding of sporadic, polygenic, undiagnosed, and rare disorders using the adequate experimental design following appropriate gene editing toolbox selection