Abstract
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.
Highlights
The development of methods for the reprogramming of terminally differentiated somatic cells to a pluripotent state has provided new opportunities to study complex diseases in a simplified and accessible system as well as generate mature cell populations for regenerative medicine-based therapies
We discuss the current state of genome editing technologies in human induced pluripotent stem cells (hiPSCs), with an emphasis on zinc-finger nuclease- (ZFN), transcription activator-like effector nuclease- (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based methods
Rouet andand colleagues in thein1990s demonstrated that double-stranded break (DSB) created by site-specific nucleases (SSNs) can be successfully repaired by or through the by site-specific nucleases (SSNs) can be successfully repaired by non-homologous end joining (NHEJ) or through the endogenous endogenous homology-mediated using an exogenously provided repair[54,55]
Summary
The development of methods for the reprogramming of terminally differentiated somatic cells (e.g., skin fibroblasts, peripheral blood mononuclear cells) to a pluripotent state has provided new opportunities to study complex diseases in a simplified and accessible system as well as generate mature cell populations for regenerative medicine-based therapies. Since the initial reports of Yamanaka and colleagues generating induced pluripotent stem cells (iPSCs) from mouse [1] and subsequently human [2] cells by the ectopic expression of four transcription factors—KLF4, OCT4, SOX2, and cMYC—several improvements to iPSC generation methods have been reported including (i) generation of hiPSCs in the absence of the oncogene cMYC [3], (ii) development of ‘foot-print free’ episomal- [4], mRNA- [5], protein- [6], and chemical-based [7] reprograming methods that do not require genomic integration of the reprogramming factors, (iii) improvements in reprogramming efficiency through the use of small molecules [8,9] and microRNAs [10], and (iv) derivation and culture of hiPSC in completely defined, xeno-free conditions [11]. Methods for HR take advantage of the endogenous DNA repair mechanisms of a cell to alter or replace a specific genomic locus with engineered transduced homologous genetic sequence Speaking, such targeting events can result in the deletion, addition, or mutation of one or a long sequence of nucleotides.
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