Although our TALENs successfully disrupted miRNA gene seeds, the resulting deletions were heterogeneous in nature as reported by others. Thus, although one can disrupt miRNA function using this method, the resulting modification is variable. It has recently been demonstrated that DNA editing can be achieved using TALENs and a single-stranded donor DNA molecule with homologous arms. striking lead improvement lv ef long term Future work should use this method to edit miRNA seed sequences in a manner that prohibits, or alters, their targeting capacity or specificity in a controlled manner. Furthermore, such an editing approach could also be used to modify miRNA-binding sites in the 39 UTRs of specific target genes, or polymorphisms within cis regulatory elements that influence miRNA expression. A recent example of such a polymorphism is found in miR-146a, which has a G/C polymorphism within in pre-miRNA sequence that reduces its expression and contributes to a predisposition to papillary thyroid cancer. Polymorphisms within the miR-155 gene have also been associated with its altered expression in human Multiple Sclerosis patients. We also found that each TALEN pair had a different functional efficiency as determined by the rate of target allele mutations. This indicates that despite following established design guidelines, additional factors are able to influence TALEN function. These may include chromatin structure, DNA modifications such as methylation, or other DNA sequence variations that influence TALEN binding dynamics. However, such determinants are presently being investigated, as this is a relatively new field of study. The capacity to deliver TALENs to precise cell types is also a challenging endeavor. Similar to other studies, we have demonstrated that transfection of cells with plasmids encoding the TALEN pair can be used to express TALENs in target cells. However, the development of viral vector systems that enable transient expression of TALENs in specific cell types is necessary for many important applications in vivo. As we continue to understand how miRNAs regulate mammalian biology, both in physiological and pathological contexts, it is becoming increasingly necessary to develop tools with the ability to specifically target and modify human miRNA genes in vivo. Based upon our findings here, TALENs make excellent candidates to achieve miRNA gene targeting and manipulation in a variety of relevant human cell types, including those with important therapeutic applications, such as stem cells, neurons and primary tumors. The pluripotency and self-renewal of embryonic stem cells are largely controlled by core pluripotency factors, Oct4, Sox2 and Nanog. They are highly expressed in ES cells and repressed during ES cell differentiation. They together bind to the promoters of many genes and activate gene expression to maintain ES cell identity and at the same time repress lineage determinants. Other transcription factors also participate in the regulation of the ES cell pluripotency and self-renewal. LIF is essential for the maintenance of mouse ES cells in the undifferentiated state. LIF binds to receptors on the cell membrane and through the parallel signaling pathways of JakSTAT3-Klf4, MAPK-Tbx3 and PI3K-Tbx3 to activate the expression of pluripotency genes. Kruppel-like factor 4 is a transcription factor that has a C2H2 zinc finger DNA binding domain at its C terminus. In Drosophila, the Kruppel protein regulates gap formation during embryonic development. In mice, the knockout of Klf4 causes neonatal lethality. In mouse ES cells, Klf4 is highly expressed in the presence of LIF, and is rapidly decreased in the absence of LIF. Klf4 and the core pluripotency factors, Oct4, Sox2 and Nanog, synergistically bind to the promoters of many genes to regulate their expression.