Two- and three-input TALE-based AND logic computation in embryonic stem cells

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations

Bacteria herald a new era of gene editing.

The demonstration that nucleases guided by bacterial RNA can disrupt human genes represents a landmark in the rapidly developing field of genome engineering

5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme

5-hydroxymethylcytosine is a new epigenetic modification deriving from the oxidation of 5-methylcytosine by the TET hydroxylase enzymes. DNA hydroxymethylation drives DNA demethylation events and is involved in the control of gene expression. Deregulation of TET enzymes causes developmental defects and is associated with pathological conditions such as cancer. Little information thus far is available on the regulation of TET activity by post-translational modifications. Here we show that TET1 protein is able to interact with PARP-1/ARTD1 enzyme and is target of both noncovalent and covalent PARylation. In particular, we have demonstrated that the noncovalent binding of ADP-ribose polymers with TET1 catalytic domain decreases TET1 hydroxylase activity while the covalent PARylation stimulates TET1 enzyme. In addition, TET1 activates PARP-1/ARTD1 independently of DNA breaks. Collectively, our results highlight a complex interplay between PARylation and TET1 which may be helpful in coordinating the multiple biological roles played by 5-hydroxymethylcytosine and TET proteins

Changes in toxin content, biomass and pigments of the dinoflagellate Alexandrium minutum during nitrogen refeeding and growth into nitrogen or phosphorus stress

Two strains oi the paralytic shellfish toxin (PST) producing dinoflagellate Alexandrium minutum Halim (highly toxic ALl V and weakly toxic AL2V) were grown in batch culture with either nitrate or phosphate as the limiting nutrient. In comparison with cells of the strain AL1V, cells of AL2V grew at a similar C-specific Tale, had a higher C/N ratio, and lower ratios of chl a/chl C2and chl a/peridinin. Neither chlorophylls flor carotenoids could be used to estimate C-biomass, N-biomass or toxin content for this organismo The toxin profile for both strains was dominated (up to 95 %) by the gonyautoxin GTX4, with smaller proportions of GTX1, GTX2 and GTX3. The Tale of toxin synthesis for both strains was greatest 1 to 2 d after the N-refeeding of N-deprived cells, with the net Tale of toxin syn- .thesis exceeding that of C-biomass and cell division by a factor of up to 4. Toxin synthesis was not enhanced by short-term P-stress. N-stress alone led to a decrease in toxin cell-I, but P-stress followed by N-stress did not result in such a decline, implicating phosphorus in the regulation of toxin metabolism. Although arginine is a majar precursor for PST synthesis, taurine, glycine, glutamine, and cell N showed similar relations to that observed for arginine with respect to toxin contento Furthermore, the mole ratio of arginine/toxin could vary by a factor of up to 5 between AL1V and AL2V at peak values of toxin cell-1, and by more than 5 within a strain when growing under different conditions. These observations suggest that the relationship between free arginine content and toxin content is complex. No explanation for the higher toxin content of AL1V is apparent, except that AL1V has a higher N-content per cell and this may be conducive to a higher Tale of synthesis of the N-rich toxins.Publicado