Application of CRISPR/Cas9 to human-induced pluripotent stem cells: from gene editing to drug discovery

Abstract

Human-induced pluripotent stem cells (hiPSCs) and CRISPR/Cas9 gene editing system represent two instruments of basic and translational research, which both allow to acquire deep insight about the molecular bases of many diseases but also to develop pharmacological research. This review is focused to draw up the latest technique of gene editing applied on hiPSCs, exploiting some of the genetic manipulation directed to the discovery of innovative therapeutic strategies. There are many expediencies provided by the use of hiPSCs, which can represent a disease model clinically relevant and predictive, with a great potential if associated to CRISPR/Cas9 technology, a gene editing tool powered by ease and precision never seen before. Here, we describe the possible applications of CRISPR/Cas9 to hiPSCs: from drug development to drug screening and from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2.

Human Embryo Models and Drug Discovery

“…Their tri-lineage differentiation potential together with their non-transformed status are obvious advantages of hPSCs over conventional immortalized human cell culture models. These inherent characteristics of hPSCs including the fact that stem cells are perfectly amenable to genetic modifications [ 34 , 35 ] make them a powerful tool for both, studies on the molecular basis of human diseases and hPDD [ 4 , 31 , 36 , 37 , 38 ]. On the other hand, as for most other cell types, traditional 2D cultivation of hPSCs and their differentiated derivatives exhibits inevitable technological limitations and conceptual biases.…”

“…In between, in a variety of studies, especially hiPSC-derived cells have been used for testing the efficacy and toxicity of different compounds [ 34 , 37 ]: hiPSC-derived cortical neurons were used to test the impact of 3838 compounds in Lesch-Nyhan disease [ 39 ] and 17,000 small molecules were tested for their anti-fibrotic effects in mesenchymal-like cells [ 40 ]. hiPSC-derived neurons, neural progenitor cells, and motor neurons have been generated to study the effects of the R33 molecule for Alzheimer’s disease, the effects of 135 compounds for Schizophrenia, and 1416 drugs for Amyotrophic lateral sclerosis, respectively [ 41 , 42 , 43 ].…”

CRISPR-Cas and Its Wide-Ranging Applications: From Human Genome Editing to Environmental Implications, Technical Limitations, Hazards and Bioethical Issues

“…In the current pandemic scenario, studies have found that CRISPR/Cas9 has potential applications to human-induced pluripotent stem cells (hiPSCs), ranging from gene therapy to the induction of the immunological response to specific virus infection, such as HIV and SARS-Cov-2 itself [ 113 ]. The potential applications of CRISPR/Cas9 and hiPSCs in antiviral response, including SARS-Cov-2 research, are centered around a testing platform meant to replicate the human lung, differentiating wild type (WT)-hiPSCs into pneumocytes type II [ 114 ], and treating them with pseudoviruses capable of replicating SARS-Cov-2 infection [ 115 ]. Although future prospects for therapeutic applications are still far from conclusive, especially as far as mutation-prone viruses such as SARS-Cov-2 are concerned, that may certainly be one way in which gene editing could be harnessed for the repression or the upregulation of genes that play a role in viral activity, in addition to the introduction of polymorphisms that could protect against or predispose to the viral infection [ 116 ].…”

Basic and Preclinical Research for Personalized Medicine

“…The implementation of iPSCs in personalized medicine has dramatically benefited from the recent advancements in DNA editing techniques, i.e., the 2020 Nobel-awarded development of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system 9 (Cas9) genome engineering tool [ 79 ]. CRISPR/Cas9-editing applied to the iPSC genome is widely exploited in translational precision medicine research for the development of novel cell-based therapeutic strategies and in the early phases of drug discovery [ 80 , 81 , 82 , 83 , 84 ]. Precisely, the artificial CRISPR system for gene editing consists of two essential components, a guide RNA (gRNA) to target complementary DNA sequences for cleavage and the CRISPR-associated protein (Cas), an RNA-guided DNA endonuclease found in Streptococcus pyogenes, which generates site-specific double-strand breaks based on the gRNA-defined sequence [ 85 , 86 ].…”