Generation of isogenic lines using CRISPR/Cas9 technology from induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cell (iPSC) technology, developed by Yamanaka in 2006, consists of rejuvenating somatic cells by introducing four reprogramming genes (OCT4, SOX2, KLF4 and C MYC). Like embryonic stem cells (ESCs), iPSCs are able to proliferate indefinitely in vitro and differentiate into derivatives of the three primary germ layers (ectoderm, mesoderm and endoderm). Due to their special features, they have been widely used in numerous studies as disease model system to study the endophenotype of the cell type affected in the disorder of interest. They have been particularly useful for diseases that are difficult to study, such as rare neurological genetic diseases. In particular, the study of neurological disorders has always been hampered by the inaccessibility of nervous system. In this regard, iPSCs offer an innovative solution, allowing personalised cell models to be generated directly from the patient's cells, opening up new perspectives for understanding and treating neurodegenerative diseases. Despite the enormous potential of iPSCs, a significant limitation is the variability of the results obtained. This heterogeneity can significantly affect the interpretation of the experimental outcome, making it difficult to establish a clear link between cellular phenotype and clinical features. In this context, it is necessary to avoid any possible variability that may be due to the experimental setup and/or and genetic background between patient-derived cells and controls. For this reason, the use of isogenic cell lines obtained by gene editing techniques represents a promising strategy to overcome major limitations due to the genetic background differences between patient and control samples. Comparing patient cells directly with their isogenic controls allows to e minimise the effects of background genetic factors. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, often referred to as 'molecular scissors', is a widely used gene-editing technology allowing DNA to be edited with unprecedented precision. This technique requires the in silico construction of a guide RNA (gRNA) containing a homologous 20 nucleotide sequence, followed by a trinucleotide (NGG) protospatial adjacent motif (PAM) in the target, and the expression of a CRISPR-associated endonuclease (Cas). Despite its versatility, the application of CRISPR/Cas9 technology to iPSCs still presents some challenges. Homologous recombination, the cellular mechanism for precisely repairing DNA breaks made by CRISPR/Cas9, appears to be less efficient in iPSCs than in other cell types. This implies that it is more difficult to obtain cell clones with the desired genetic modification, slowing down the development of accurate cell models for studying genetic diseases. Genome editing in iPSCs is further impaired by their tendency to undergo programmed cell death when cultured as single cells. This requirement, which is essential for the selection of genetically modified clones, provides a significant challenge for the manipulation of these cells. To overcome the low efficiency of homologous recombination and to facilitate the identification of genetically modified clones, selectable reporter systems or markers, such as green fluorescent protein (GFP) or resistance to a specific antibiotic, are often used. These systems allow direct 9 visualisation or selection of the cells that have undergone the editing event. Transposons such as piggyBac can be used to safely remove the unnecessary constructs. However, it is important to note that this system can cause off-target effects. Therefore, optimisation of the experimental protocols is crucial to ensure the reliability of the results. The ability of CRISPR/Cas9 to make precise cuts in DNA, combined with the efficiency and specificity of piggyBac to insert and remove DNA sequences, has enabled targeted genetic manipulation of a wide range of organisms, from cells in vitro to animal models. This powerful synergy has opened up new perspectives in biomedical research, facilitating the generation of reliable cellular models for the study of genetic diseases. The use of an in vitro model generated combining CRISPR/Cas9 technology with iPSCs offers unique opportunities to study the pathophysiological mechanisms underlying rare paediatric neurodegenerative and neurodevelopmental diseases.