Journal: Journal of Bacteriology

Article Title: A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus

doi: 10.1128/jb.00335-23

Figure Lengend Snippet: Overview of the dual-plasmid CRISPR/Cas9 workflow. ( A ) Sth1 Cas9 mediates DSBs at the target site, which leads to either bacterial death in the absence of DNA repair or inaccurate indels upon nontemplated DNA repair. Created with BioRender. ( B ) M. abscessus was first transformed with the inducible Cas9 on an integrative plasmid pDN-Sth1Cas9 containing the tetracycline repressor TetR and resistance selection marker Kan R . This Cas9-background strain was then transformed with the sgRNA cassette on a mCherry-expressing plasmid pDN-Cherry-sgRNA containing the resistance selection marker Hyg R . The induction of the CRISPR system leads to gene disruption and loss of function. Finally, curing the mCherry-sgRNA plasmid creates the edited strain still carrying the inducible Cas9 to allow the subsequent introduction of additional sgRNA cassettes.

Article Snippet: The resulting plasmid pDN-Sth1Cas9 contains genes for the AHT-inducible Sth1 Cas9, L5 integrase for integration into the attB site, tetracycline repressor TetR, and resistance selection marker Kan R . pCHERRY3 (Addgene plasmid #24659; http://n2t.net/addgene :24659; RRID: Addgene_24659; gift from Tanya Parish) was used as the template for the construction of the sgRNA-containing plasmid ( ).

Techniques: Plasmid Preparation, CRISPR, Transformation Assay, Selection, Marker, Expressing, Disruption

Journal: Science Advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: ( A ) Position of the four 300–amino acid fragments of NFAT5 tested in (B) to (D). ( B ) Recruitment of endogenous BRD4 (red) to a TetO array in U2OS cells by EGFP-TetR DBD (green) fused to the four fragments of NFAT5 (see fig. S12A). Insets show a magnified view of the TetO array, visualized as a single dot of EGFP fluorescence. Enrichment of BRD4 in the EGFP-marked TetO array is plotted on the right for individual cells, with the mean indicated. Scale bars, 10 μm. ( C ) Condensate formation by hemagglutinin-tagged NFAT5 fragments in HEK293T cells ( n > 25, median indicated). ( D ) Transactivation capacity of NFAT5 fragments ( n = 3, bars show mean) or the VP16 AD (as a control) using the reporter assay shown in . ( E ) A model for hypertonic and ionic stress adaptation. The IDR in WNK1 and PLD in NFAT5 each sense specific chemical properties of the intracellular environment. In response to hypertonic stress, the rapid loss of cell volume leads to an increase in macromolecular crowding, which activates the crowding sensor kinase WNK1 (but not NFAT5) . Through a kinase cascade, WNK1 activates transporters that increase intracellular ion concentrations, allowing cytoplasmic rehydration and volume recovery at the expense of elevated ionic strength. If persistent, this increase in ionic strength is the trigger for NFAT5 activation, leading to a transcriptional response that exchanges these ions for osmolytes. We speculate that NFAT5 has evolved to sense and facilitate adaptation to diverse ionic stressors (even those, like NH 4 OAc, that do not cause hypertonic stress). Statistics: Statistical significance was determined by a Kruskal-Wallis test, Dunn’s multiple comparisons [(B) and (C)], or a two-way ANOVA with Sidak’s multiple comparisons test (D). **** P < 0.0001 and ** P < 0.01. See also figs. S12 and S13.

Article Snippet: As a control, the constitutive transcriptional activator VP16 was transfected using pEGFP-TetR-NLS-VP16 (Addgene plasmid #103834).

Techniques: Fluorescence, Control, Reporter Assay, Activation Assay