CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

Abstract

Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology-and particularly clustered regularly interspaced short palindromic repeats (CRISPR)-will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.

Keywords: Clustered regularly interspaced short palindromic repeats; Clustered regularly interspaced short palindromic repeats-Cas9; Gene editing; Genetic therapy; Induced pluripotent stem cells.

Figure 1.

Timeline of technological progression of clustered regularly interspaced short palindromic repeats (CRISPR) and its application in model organisms. Key developments are shown and major breakthroughs are highlighted in white boxes. While the CRISPR story starts in 1987, the name was coined in 2000, and CRISPR’s role in adaptive immune system was demonstrated in 2007. A key insight in 2012 that CRISPR-associated nuclease 9 (Cas9) is an RNA-guided DNA endonuclease led to an explosion of papers related to CRISPR gene-editing technology. From 2013, CRISPR was successfully applied in modification of genes in humans and other various organisms [4-36]. sgRNA, single guide RNA; P. falciparum, Plasmodium falciparum; X. tropicalis, Xenopus tropicalis; C. elegans, Caenorhabditis elegans; A. thaliana, Arabidopsis thaliana; D. melanogaster, Drosophila melanogaster; tracrRNA, trans-acting CRISPR RNA; crRNA, CRISPR RNA; E. coli, Escherichia coli.

Figure 2.

Simplified mechanism of microbial adaptive immune system using clustered regularly interspaced short palindromic repeats (CRISPR). Upon entry of foreign DNA into bacteria, CRISPR-associated (Cas) enzymes acquire new spacers from the exogenous sequence and integrate this spacer unit into the leader end of CRISPR locus within bacterial genome. The transcript of CRISPR array is further processed, and when another corresponding invasion occurs this mature CRISPR RNA (crRNA) act as a guide by Cas complex to degrade matching DNA. The detailed mechanisms of each type of CRISPR systems vary slightly. (A) Acquisition. (B) crRNA biogenesis. (C) Interference.

Figure 3.

Overview of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) gene editing from target selection and guide design to validation. (A) Select gene of interest and design guide RNA. (B) Base pairing of sgRNA: genomic DNA. (C) Detection of PAM by Cas and cleavage of gene of interest by Cas domains HNH and RuvC. (D) Formation of nuclease-induced double strand breaks (DSB). (E) Validation of gene editing. sgRNA, single guide RNA; crRNA, CRISPR RNA; tracrRNA, trans-acting CRISPR RNA; PAM, proto-spacer adjacent motif; NHEJ, nonhomologous end joining; HDR, homology-directed repair.

Figure 4.

Overview of gene editing and its applications. Genetic defects can be corrected via gene editing with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR) system. When double-strand breaks occur, the lesion can be corrected by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR) pathways. Arising from this technique, gene editing can be applied in various fields of research and biotechnology. sgRNA, single guide RNA; PAM, proto-spacer adjacent motif; DMD, Duchenne muscular dystrophy; HIV, human immunodeficiency virus; HBV, hepatitis B virus; CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 5.

Generation of edited induced pluripotent stem cells (iPSCs) and clinical applications thereof. Somatic cells isolated from a normal person or patient are reprogrammed into iPSCs. Normal sequence can be disrupted or genetic defects can be corrected via gene editing. iPSCs with edited modifications are differentiated into various target cells for disease modeling, which can provide a useful channel for precision therapy and drug screening. ZFN, zinc finger nuclease; TALEN, transcription activator-like effector nuclease; CRISPR, clustered regularly interspaced short palindromic repeats.