Specific nucleases (SNs), including ZFNs, TALENs, and CRISPR (clustered regularly interspaced palindromic repeats), are powerful tools for genome editing (GE). These tools have achieved efficient gene repair and gene disruption of human primary cells. However, their efficiency and safety must be improved before translation into clinic. In particular, one of the main hurdles of GE technology is the delivery of the different components into the nucleus of target cells. Successful gene editing must be able to deliver the SNs and/or the donor DNA into a large number of target cells in order to have a therapeutic benefit. In addition, the delivery must be nontoxic and the SNs must be innocuous to the target cells. In this chapter, we will summarize the different ways to deliver SNs and donor DNA.
Part of the book: Modern Tools for Genetic Engineering
Human pluripotent stem cells (PSCs) in the form of human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are capable of growing indefinitely in vitro, maintaining their capacity to differentiate into the three primary germ layers: mesoderm, endoderm and ectoderm. Different protocols have been developed to differentiate PSCs into almost any cellular type with different degree of success. This technology has allowed scientists to use patient‐derived iPSCs to study the physiopathology of the disease by analyzing the phenotype of the cells derived from these iPSCs. However, control iPSCs obtained from healthy individuals will always have different genomic environment than patient's iPSCs, making it difficult the interpretation of the cells phenotype. The recent appearance of specific nucleases [zinc‐finger nucleases (ZFNs), the transcription activator‐like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)] has made it possible to edit the genome of PSCs. We can now generate syngeneic hESCs or iPSCs harboring the desired mutation and comparing the emerging cells with those derived from genetically identical PSCs that will differ only in the mutated gene. In this chapter, we summarize the progress made in this field and discuss the different approaches that have been used recently for the generation of syngeneic human pluripotent cellular models for different pathologies.
Part of the book: Pluripotent Stem Cells