Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

The antitrypanosomal drug melarsoprol competitively inhibits thiamin uptake in mouse neuroblastoma cells

Melarsoprol is the main drug used for the treatment of late-stage sleeping sickness, although it causes severe side-effects such as encephalopathy and polyneuropathy leading to death in some patients. Recent data suggest that melarsoprol and its active metabolite melarsenoxide interfere with thiamin transport and metabolism in E. coli and yeast, but there are no data concerning their possible effects on thiamin metabolism in mammalian cells. We tested both drugs on thiamin transport in cultured mouse neuroblastoma cells using C-14-labeled thiamin. Melarsoprol, competitively inhibits high-affinity thiamin transport in mouse neuroblastoma cells with a K-i of 44 mu mol/L. However, the active compound melarsenoxide has no inhibitory effect. This suggests that the side effects of melarsoprol treatment are unlikely to be due to inhibition of thiamin transport by melarsenoxide, its main metabolite in the brain

Pig tissues express a catalytically inefficient 25-kDa thiamine triphosphatase: Insight in the catalytic mechanisms of this enzyme

peer reviewedThiamine triphosphate (ThTP) is found in most organisms and may be an intracellular signal molecule produced in response to stress. We have recently cloned the cDNA coding for a highly specific mammalian 25-kDa thiamine triphosphatase. The enzyme was active in all mammalian species studied except pig, although the corresponding mRNA was present. In order to determine whether the very low ThTPase activity in pig tissues is due to the absence of the protein or to a lack of catalytic efficiency, we expressed human and pig ThTPase in E. coli as GST fusion proteins. The purified recombinant pig GST-ThTPase was found to be 2-3 orders of magnitude less active than human GST-ThTPase. Using site-directed mutagenesis, we show that, in particular, the change of Glu85 to lysine is responsible for decreased solubility and catalytic activity of the pig enzyme. Immunohistochemical studies revealed a distribution of the protein in pig brain very similar to the one reported in rodent brain. Thus, our results suggest that a 25-kDa protein homologous to hThTPase but practically devoid of enzyme activity is expressed in pig tissues. This raises the possibility that this protein may play a physiological role other than ThTP hydrolysis

Recombination-based complementation of FAN1 knockout cells.

<p>(<b>A</b>) U2OS SEC-C FAN1^−/− cells were transfected or not with the plasmid pOG44 expressing the Flp recombinase. After 5 passages under zeocin selection, cell extracts were subjected to western blotting with anti-Flag antibodies to check for the elimination of the Cas9 ORF. (<b>B</b>) Lysates from Flp-In T-REx U2OS FAN1^−/− cells transfected or not with the plasmids pOG44 and pCDNA5-FRT-GFP-FAN1 were subjected to western blotting with the indicated antibodies. (<b>C</b>) Clonogenic survival analysis of the cells U2OS FAN1^+/+ (clone 2) U2OS FAN1^−/− (Clone 1) and U2OS FAN1^−/− + FAN1 (Clone 1 complemented) after exposure to MMC. For each genotype, cell viability of untreated cells is defined as 100%. Data are represented as mean ± SEM, n =  Experimental significance was calculated using a unpaired T-test correct using Holm-Sidak method; *, p<0.01.</p

Fast PCR-based generation of sgRNA-encoding plasmids.

<p>(<b>A</b>) Table showing primers used to generate the sgRNA plasmids (pEsgRNA) for five different human ORFs using PCR-based insertion mutagenesis: NM_152486, NM_015658, NM_014967 (FAN1), NM_001009608 and NM_024631. (<b>B</b>) gRNA sequence transcribed from the U6 promoter. (<b>C</b>) sgRNA plasmids purified from bacteria were BamHI digested and separated using 1% agarose gel in TAE buffer. The arrow indicates the 300 bp BamH1 digest fragment.</p

Characterizing arrhythmia using machine learning analysis of Ca2+cycling in human cardiomyocytes

10.1016/j.stemcr.2022.06.005STEM CELL REPORTS1781810-182

Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2

USP45 deubiquitylase controls ERCC1-XPF endonuclease-mediated DNA damage responses

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair