ABSTRACT
In this issue of Cell, Nuñez et al. develop CRISPRoff, a programmable epigenetic memory writer capable of establishing specific gene silencing programs that are stably maintained across cell division and differentiation. The singular dCas9 fusion offers a simple, reliable, and general tool for genome-wide screens, multiplexed editing, and potential therapeutics.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Epigenomics , Promoter Regions, Genetic , WritingABSTRACT
Genome-wide studies have defined cell type-specific patterns of DNA methylation that are important for regulating gene expression in both normal development and disease. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator-like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.
Subject(s)
DNA Methylation/genetics , DNA-Binding Proteins/genetics , Gene Targeting/methods , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins/genetics , Recombinant Fusion Proteins/genetics , Transcription Factors/genetics , Up-Regulation/genetics , Humans , Mixed Function OxygenasesABSTRACT
Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.
Subject(s)
Genetic Engineering/methods , MicroRNAs/genetics , Transcription Factors/genetics , Transcriptional Activation , Vascular Endothelial Growth Factor A/genetics , Binding Sites , Cell Culture Techniques , Fibroblasts/metabolism , HEK293 Cells , Humans , Plasmids , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Tandem Repeat Sequences , Transfection , Xanthomonas , Zinc Fingers/geneticsABSTRACT
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.