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Tsai, H.-H.

Publications and source records attributed to Tsai, H.-H..

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pH-Dependent Transcriptional Profile Changes in Iron-Deficient Arabidopsis Roots

Iron is an essential element for plants and abundantly present in most mineral soils. The mobility of iron is, however, dependent on the redox potential and hydrogen activity (pH) of the soil, factors that may limit its availability to plants in particular at alkaline pHs. Iron deficiency triggers pronounced changes in the transcriptional profile of plants, inducing processes that aid in the acquisition, uptake, and translocation of iron. How ambient pH impact the transcriptional iron deficiency response has not yet been elucidated in detail. Here, we provide an RNA-seq data set that catalogs global gene expression changes of iron-deficient plants grown at either optimal (5.5) or high (7.0) pH. A suite of 857 genes changed significantly and more than twofold in expression; only 54 genes of this suite were also differentially expressed between iron-deficient and iron-sufficient plants grown at pH 5.5. Among the high pH-responsive genes, 186 were earlier shown to be responsive to short-term transfer to low pH, 91 genes of this subset were anti-directionally regulated by high and low pH. The latter subset contained genes involved in cell wall organization, auxin homeostasis, and potential hubs of yet undefined signaling circuits. Growing iron-deficient plants at high pH also modulated the transcriptional iron deficiency response observed at pH 5.5 by compromising the enzymatic reduction of ferric chelates and favoring the production of iron-mobilizing coumarins. It is concluded that ambient pH is an important determinant of global gene expression which tunes iron acquisition to the prevailing edaphic conditions.

plant biology

Phenotypic screening using synthetic CRISPR gRNAs reveals pro-regenerative genes in spinal cord injury

Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest. HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active - in vivo pre-screening allows rapid assessment of CRISPR gRNA activity - Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration - tgfb1a promotes spinal regeneration by controlling inflammation

neuroscience