Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation.

Joseph Collin,Inmaculada Moreno-Gimeno,Stefan Przyborski,Carla B Mellough,Birthe Dorgau,Majlinda Lako

doi:10.1002/stem.2240

Abstract

The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells 2016;34:311–321

Highlights

Zinc finger nucleases (ZFNs) are designer nucleases which can be engineered to target a specific DNA sequence, offering huge potential for genetically modifying cells with complex genomes, such as mammalian cells [1]
We mutated the stop codon of Cone-Rod Homeobox (CRX) introduced in the targeting cassette, which was followed by the eGFP sequence (Fig. 1A; Supporting Information Fig. 2), resulting in a CRX-green fluorescent protein (GFP) fusion upon ZFN pair cutting in the 30UTR of CRX and insertion of the targeting cassette via homologous recombination
Quantitative polymerase chain reaction (PCR) based copy number analysis of GFP revealed the presence of two copies of GFP (Fig. 1C), corroborating the PCR and sequencing data and suggesting that no additional copies of GFP were present in the genome due to nontargeted insertion of the donor cassette

Summary

Introduction

Zinc finger nucleases (ZFNs) are designer nucleases which can be engineered to target a specific DNA sequence, offering huge potential for genetically modifying cells with complex genomes, such as mammalian cells [1]. ZFNs are comprised of a DNA binding domain of zinc finger protein motifs (N terminal) fused to the FokI endonuclease domain (C terminal), a DNA cleaving domain which operates upon dimerization [1]. ZFNs are designed to work as a pair; upon binding to target sites on opposing strands, they act as a heterodimer and cleave both strands of the DNA. While only transient expression of ZFNs is required over a brief period of in vitro culture, the resulting genetic manipulation is present for the life of the cell, avoiding the need for continued expression of a foreign transgene. The potential for gene targeting and editing in various complex genomes is considerable and has been realized in multiple organisms [1]

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