Genome-editing tools for stem cell biology

Abstract

Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR).

Figure 1

Application of genome editing in molecular medicine (gene therapy, disease modeling). iPCSs could be generated from somatic cells of the patient with monogenic diseases for correction, differentiation into cell types suitable for therapy and transplantation into a patient to restore the function

Figure 2

ZFs, TALEs and CRISPR/Cas9 systems for genome editing and gene expression manipulation. ZFP, zinc-finger protein; FD, functional domain; TFs, transcription factors; dCas9, dead Cas9 nuclease; SL1-3, stem loop 1-3; PAM, protospacer adjacent motif. (a) Schematic representation of the ZFN (zinc-finger nuclease) system for genome editing. It consists of a zinc-finger DNA-binding domain and a nuclease domain of the FokI endonuclease. (b) Site-specific ZF-TFs can either activate or repress gene expression depending on their functional domains (FD). (c) Schematic representation of the TALENs system for genome editing. It consists of a TALEs DNA-binding domain and a nuclease domain of the FokI. XX- RVDs, repeat variable di-residues. (d) TALE-TFs can also either activate or repress transcription. (e) Schematic representation of the LITE-system (light-inducible transcriptional effectors) consists of DNA-binding TALE domain with the photosensitive protein CRY2 (TALE:CRY2) and CIB1 (interaction partner with CRY2), coupled with the desired effector (complex CIBI: effector). In the absence of light TALE:CRY2 are joined to the promoter region of a target gene, whereas a complex CIB1: effector remains free (OFF) (see the text). NLS, nuclear localization signal. (f) LITE system after light illumination, which confers conformational changes into the CRY2 protein, which subsequently recruits the CIB1:effector complex and a number of transcription factors to the promoter region of the target gene to activate transcription (ON) (see the text). (g) Schematic representation of the CRISPR/Cas9 system for genome editing, which consists of Cas9 nuclease domain and joined crRNA and tracrRNA for directing the Cas9 nuclease to the target site. The target site is indicated by scissors. PAM is shown inside the circles. (h) CRISPR/Cas9 system includes Cas9 nuclease domain and sgRNA for directing the Cas9 nuclease to the target site; (i) Site-specific binding of dCas9 with sgRNA can inhibit the interaction of TFs with a promoter region causing gene repression; (k) Site-specific binding dCas9:sgRNA fused to FD facilitates transcription

Figure 3

Structure of Oct4 upstream promoter region. (a) Schematic representation of the Oct4 upstream region of the human promoters. CR1-4 denote Conservative Regions in the promoter of Oct4 gene (see the text). Conserved sequences are shown inside the boxes. Their locations relative to the start site are indicated below. Known transcription factors that bind these CRs are indicated. (b) Shown is the upstream region in the promoter of Oct4 gene. Specific DE and PE sites with respect to the CRs are indicated. Green arrow denotes the direction of Oct4 gene transcription