Custom-designed nucleases (CDNs) greatly facilitate genetic engineering by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, specific modifications can be introduced around the breakage site during its repair by two major DNA damage repair (DDR) mechanisms: the dominant but error-prone nonhomologous end joining (NHEJ) pathway, and the less-frequent but precise homologous recombination (HR) pathway. Here we describe ObLiGaRe, a new method for site-specific gene insertions that uses the efficient NHEJ pathway and acts independently of HR. This method is applicable with both zinc finger nucleases (ZFNs) and Tale nucleases (TALENs), and has enabled us to insert a 15-kb inducible gene expression cassette at a defined locus in human cell lines. In addition, our experiments have revealed the previously underestimated error-free nature of NHEJ and provided new tools to further characterize this pathway under physiological and pathological conditions.

Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels. TMEM203 protein was localized to the ER and found associated with a number of ER proteins which regulate ER calcium entry and efflux. Mouse Embryonic Fibroblasts (MEFs) derived from Tmem203 deficient mice had reduced ER calcium stores and altered calcium homeostasis. Tmem203 deficient mice were viable though male knockout mice were infertile and exhibited a severe block in spermiogenesis and spermiation. Expression profiling studies showed significant alternations in expression of calcium channels and pumps in testes and concurrently Tmem203 deficient spermatocytes demonstrated significantly altered calcium handling. Thus Tmem203 is an evolutionarily conserved regulator of cellular calcium homeostasis, is required for spermatogenesis and provides a causal link between intracellular calcium regulation and spermiogenesis.