alexa Application of CRISPR/cas9-Directed Homologous Recombination to the Generation of Human Tumor Cells with Conditional Knockout of an X-Linked MicroRNA Locus

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Application of CRISPR/cas9-Directed Homologous Recombination to the Generation of Human Tumor Cells with Conditional Knockout of an X-Linked MicroRNA Locus
MicroRNAs (miRNAs) are small noncoding RNAs that exert important cellular functions by repressing post-transcriptional gene expression through binding to target mRNAs . More than 60% of all human genes are predicted to be regulated by a total of over 2.000 mature miRNAs. Some miRNAs are expressed in virtually all cell types, whereas others are highly tissue-specific with a distinct function depending on cell type or organ. miRNAs play key roles in most biological processes, including cell division and death, cellular metabolism, intracellular signaling, immunity and cell movement. Not surprisingly, disregulation of miRNA expression has been functionally linked to various pathological and occasionally malignant outcomes [2] Specific miRNA expression patterns, which have been associated with particular diseases, hold great prognostic value. In cancer, oncogenic miRNAs as well as tumor suppressor miRNAs have been identified, and therapeutic strategies directed against such cancer-related miRNA are being considered. The physiological roles of most miRNAs still need to be deciphered. Therefore, tools allowing manipulation of miRNA activity are required. Gain-of-function by over-expression of the miRNA is relatively easy, and can be achieved by transfection of synthetic microRNAs or by enforcing expression of primary miRNA transcripts . Lossof- function analyses are less obvious. miRNA inhibitors based on synthetic antisense molecules or miRNA sponges, which act by binding and sequestering miRNAs away from their natural targets, are available . However, such inhibitors have some disadvantages, including incomplete masking of miRNA function, and uncontrolled off-target effects.
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