The deactivated CRISPR/Cas9 (dCas9) is now the most widely-used gene activators. However, the current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, CRISPR-assisted trans enhancer, for activating gene expression in high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, a CMV enhancer DNA was recruited to target gene in trans by two systems, dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GLA4/sgRNA-UAS-CMV. The former recruited trans enhancer by the annealing between two short complementary oligonucleotides at the ends of sgRNA and trans enhancer. The latter recruited trans enhancer by the binding between GLA4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter gene and variant endogenous genes in various cells, with much higher activation efficiency than the current dCas9 activators.
All data generated or analysed during this study are included in the manuscript and supporting files.
- Jinke Wang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Irwin Davidson, Institut de Génétique et de Biologie Moléculaire et Cellulaire, France
© 2019, Xu et al.
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An imbalance of the gut microbiota, termed dysbiosis, has a substantial impact on host physiology. However, the mechanism by which host deals with gut dysbiosis to maintain fitness remains largely unknown. In Caenorhabditis elegans, Escherichia coli, which is its bacterial diet, proliferates in its intestinal lumen during aging. Here, we demonstrate that progressive intestinal proliferation of E. coli activates the transcription factor DAF-16, which is required for maintenance of longevity and organismal fitness in worms with age. DAF-16 up-regulates two lysozymes lys-7 and lys-8, thus limiting the bacterial accumulation in the gut of worms during aging. During dysbiosis, the levels of indole produced by E. coli are increased in worms. Indole is involved in the activation of DAF-16 by TRPA-1 in neurons of worms. Our finding demonstrates that indole functions as a microbial signal of gut dysbiosis to promote fitness of the host.
The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.