Abstract
MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes. Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes ex vivo using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b. This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.
Highlights
MicroRNAs are small, single-stranded RNAs that have been highly conserved during evolution and function by repressing target gene expression. miRNAs have recently emerged as critical modulators of gene expression networks in mammals, and their impaired expression or function has been linked to a variety of human diseases [1]
Our analysis was carried out using Transcription Activator-Like Effectors (TALEs)-NT software, and followed the parameters described in our Materials and Methods
All of the Tal-Effector Nucleases (TALENs) pairs caused at least some deletions that disrupted the seed sequences of each respective miRNA, even if their binding sites did not flank, but were near, the seed. These results indicate that TALENs can be routinely used to disrupt human miRNA seed sequences, and that the design parameters consistently allow for successful targeting of a DNA sequence as small as an 8-nucleotide miRNA seed found in specific DNA locations and contexts
Summary
MicroRNAs are small, single-stranded RNAs that have been highly conserved during evolution and function by repressing target gene expression. miRNAs have recently emerged as critical modulators of gene expression networks in mammals, and their impaired expression or function has been linked to a variety of human diseases [1]. MiRNAs can participate in the formation of induced pluripotent stem (iPS) cells [5], which hold significant promise in the field of regenerative medicine Despite these important and clinically significant roles for miRNAs, our ability to manipulate miRNA expression and function in human cells remains a challenging task. The fully processed miRNA is loaded into RISC and mediates target gene repression by directing the RISC complex to specific mRNA 39 UTRs containing cognate binding sites for the miRNA. This interaction between the miRNA and 39 UTR is dependent upon a 6–8 nucleotide sequence found in the 59 end of the miRNA called the ‘‘seed’’ sequence. MiRNAs can be modulated by controlling their expression levels or by disrupting their seed:target interaction
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