Exploiting CRISPR Cas9 in Three-Dimensional Stem Cell Cultures to Model Disease

Abstract

Three-dimensional (3D) cell culture methods have been widely used on a range of cell types, including stem cells to modulate precisely the cellular biophysical and biochemical microenvironment and control various cell signaling cues. As a result, more in vivo -like microenvironments are recapitulated, particularly through the formation of multicellular spheroids and organoids, which may yield more valid mechanisms of disease. Recently, genome-engineering tools such as CRISPR Cas9 have expanded the repertoire of techniques to control gene expression, which complements external signaling cues with intracellular control elements. As a result, the combination of CRISPR Cas9 and 3D cell culture methods enhance our understanding of the molecular mechanisms underpinning several disease phenotypes and may lead to developing new therapeutics that may advance more quickly and effectively into clinical candidates. In addition, using CRISPR Cas9 tools to rescue genes brings us one step closer to its use as a gene therapy tool for various degenerative diseases. Herein, we provide an overview of bridging of CRISPR Cas9 genome editing with 3D spheroid and organoid cell culture to better understand disease progression in both patient and non-patient derived cells, and we address potential remaining gaps that must be overcome to gain widespread use.

Historical evolution of spheroids and organoids, and possibilities of use in life sciences and medicine

“…Since it is possible to derive organoids from patients, resulting in patient-specific cultures, and that it is possible to genetically manipulate the culture by using, as an example, CRISP system (Clustered Regularly Interspaced Short Palindromic Repeats), it is expected that soon it will be possible to observe more cases in which 3D cultivated cells are used in regenerative medicine, including genetic manipulation for diseases to which there is no effective treatment nor cure. [116,117] This possibility will enable researchers and medical workers to join forces and elaborate specific treatments for specific patients. [118] 3D cultures might help us understand drug applicability, dosage establishment, including possibilities of investigating cellular damage and toxicity prior to production and distribution.…”

3D Printed Bioconstructs: Regenerative Modulation for Genetic Expression

“…[64] This further helps in cleavage of DNA at the targeted site. Further advancements in technologies like CRISPR/Cas9 system that is used in genome engineering and its incorporation into 3DP applications may emerge as a robust methods for gene therapy [65]. Similarly, mobile molecule mRNA printers or 'RNA microfactories' are said to be developed by Tesla Inc. in order to build mRNA-based vaccines for COVID-19.…”

New Modalities of 3D Pluripotent Stem Cell-Based Assays in Cardiovascular Toxicity

“…Finally, by combining iPSC and gene editing technologies (i.e. CRISPR/Cas9) one can create cells with a well-defined genetic background ( Boretto et al, 2019 ; Gopal et al, 2020 ; Sacchetto et al, 2020 ). Human iPSC cultures can remain genetically and phenotypically stable for long periods, permitting us to monitor time-dependent processes during the maintenance ( Liu et al, 2020 ).…”