Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest

SMDT1 variants impair EMRE-mediated mitochondrial calcium uptake in patients with muscle involvement

Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca2+ uptake

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Organic anion transporter 1 and 3 influence cellular energy metabolism in renal proximal tubule cells

Organic anion transporter (OAT) 1 and 3 are, besides uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells

Organic anion transporter (OAT) 1 and 3 are, besides uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype

Genetic variants in the LAMA5 gene in pediatric nephrotic syndrome.

Nephrotic syndrome (NS), a chronic kidney disease, is characterized by significant loss of protein in the urine causing hypoalbuminemia and edema. In general, ∼15% of childhood-onset cases do not respond to steroid therapy and are classified as steroid-resistant NS (SRNS). In ∼30% of cases with SRNS, a causative mutation can be detected in one of 44 monogenic SRNS genes. The gene LAMA5 encodes laminin-α5, an essential component of the glomerular basement membrane. Mice with a hypomorphic mutation in the orthologous gene Lama5 develop proteinuria and hematuria

Deep sequencing reveals high mutagenesis rates for acute multi-sgRNA CRISPR/Cas9 KO of <i>osgep</i> and <i>tprkb</i>.

<p>(<b>a</b>) Mutation rates per sgRNA (i.e. likelihood of generating at least one mutation on each allele), given hypothetical mutation rates for 3 different sgRNAs. Note that the achieved likelihood for occurrence of at least one mutation per allele is high (92.16%), even though mutation rates for each sgRNA are moderate (50–80%). (<b>b-e</b>) Deep sequencing reveals high mutagenesis rates for most sgRNAs and sgRNA dependent frameshift rates. For each gene, deep sequencing data of 96 larvae at 48 hpf were analyzed individually using the tool CRISPResso [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191503#pone.0191503.ref024" target="_blank">24</a>]. (<b>b</b>) For <i>osgep</i>, median mutagenesis rate was 69.3% for sgRNA1, 93.0% for sgRNA2, and 79.6% for sgRNA3. (<b>c</b>) For all mutated <i>osgep</i> alleles, the median fractions of frame shifts were 56.7% for sgRNA1, 44.7% for sgRNA2, and 61.0% for sgRNA3. (<b>d</b>) For <i>tprkb</i>, median mutagenesis rate was 100% for sgRNA1, 92.3% for sgRNA2, and 41.0% for sgRNA5. (<b>e</b>) For all mutated <i>tprkb</i> alleles, the median fractions of frame shifts were 44.3% for sgRNA1, 81% for sgRNA2, and 52.8% for sgRNA5.</p

Likelihoods for at least one mutation on each allele and at least one frameshift mutation on each allele are clearly improved in acute multi-sgRNA CRISPR/Cas9 KO of <i>osgep</i> and <i>tprkb</i>.

<p><b>(a-b)</b> The likelihood is shown of generating at least one mutation on each allele <i>P(M)</i> (<b>a</b>) and of generating at least one frameshift mutation on each allele <i>P(F)</i> (<b>b</b>), where <i>M</i> = at least one mutation on each allele, <i>F</i> = at least one frameshift mutation on each allele, <i>q</i> = probability of no mutation, <i>r</i> = specific sgRNA, <i>im</i> = fraction of in-frame mutations of all mutations, <i>nm</i> = fraction of non-coding mutations of all mutations. (<b>c-h</b>) The likelihoods of at least 1 mutation on each allele and at least 1 frameshift mutation on each allele were calculated based on observed mutagenesis and frameshift rates for 96 individual fish per gene according to the equation in (<b>a</b>) and (<b>b</b>). (<b>c</b>) The individual analysis for <i>osgep</i> revealed a median <i>P(M)</i> of 48.1% for sgRNA1, 86.6% for sgRNA2, and 63.4% for sgRNA3. (<b>d</b>) The median <i>P(F)</i> was 15.1% for sgRNA1, 14.6% for sgRNA2, and 18.5% for sgRNA3. (<b>e</b>) For the pooled <i>osgep</i> sgRNAs, the median <i>P(M)</i> was 99.3%, and 64.2% for <i>P(F)</i>. (<b>f</b>) The individual analysis for <i>tprkb</i> revealed a median <i>P(M)</i> of 100% for sgRNA1, 84.7% for sgRNA2, and 16.9% for sgRNA5. (<b>g</b>) The median <i>P(F)</i> was 15.5% for sgRNA1, 52.2% for sgRNA2, and 4.1% for sgRNA5. (<b>h</b>) For the pooled <i>tprkb</i> sgRNAs, the median <i>P(M)</i> was 100%, and 78.8% for <i>P(F)</i>.</p