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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.

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.

Background The spinal intrathecal space, characterized by a complex three‐dimensional (3D) fluid‐filled geometry with varying levels of anatomic intricacy, plays a pivotal role in drug administration strategies targeting the central nervous system (CNS). Lumbar injections into this space represent a clinically used approach for delivering therapeutics directly to the brain, bypassing critical barriers such as the blood‐brain and blood‐cerebrospinal fluid barriers. A nuanced understanding of CSF dynamics is vital for comprehending physiological states of the CNS for drug delivery. Method The CSF’s dynamic nature enables the dispersion of solutes throughout the brain and ventricular system. In order to optimize CSF drug delivery strategies, a profound comprehension of the intrathecal anatomy coupled with insights into the physiology of CSF generation, absorption, and flow, is imperative. The application of quantitative modeling tools, such as Computational Fluid Dynamics (CFD), yields critical insights into the impact of CSF fluid dynamics on drug dispersion and efficiency. CFD modeling provides a granular analysis of CSF flow field, surpassing the capabilities of Magnetic Resonance Imaging (MRI) or invasive methodologies. Result Eli Lilly’s initiative in developing a CSF model aimed to predict drug distribution patterns mediated by cerebrospinal flow within the intrathecal space. This model sought to simulate prototypical CSF flow to ascertain the time required for a specified drug mass to reach the brain following lumbar intrathecal injection. Additionally, it aimed to assess the subsequent distribution of the drug within the brain, across a diverse patient demographic. This approach is instrumental in fine‐tuning drug delivery protocols, customizing delivery methods to particular drugs, avoiding off‐target drug effects, and ensuring maximum efficiency and efficacy for direct CNS treatments. Conclusion Through this computational methodology, we have demonstrated the feasibility of obtaining validation data for the CFD simulations using several imaging methodologies and techniques. Furthermore, we have been able to build these complex models and use them to inform in vivo studies. We have also been able to demonstrate that it is possible to build comparable NHP and human models from the fluid dynamics perspective.

Declining tissue nicotinamide adenine dinucleotide (NAD) levels are linked to ageing and its associated diseases. However, the mechanism for this decline is unclear. Here, we show that pro-inflammatory M1-like macrophages, but not naive or M2 macrophages, accumulate in metabolic tissues, including visceral white adipose tissue and liver, during ageing and acute responses to inflammation. These M1-like macrophages express high levels of the NAD-consuming enzyme CD38 and have enhanced CD38-dependent NADase activity, thereby reducing tissue NAD levels. We also find that senescent cells progressively accumulate in visceral white adipose tissue and liver during ageing and that inflammatory cytokines secreted by senescent cells (the senescence-associated secretory phenotype, SASP) induce macrophages to proliferate and express CD38. These results uncover a new causal link among resident tissue macrophages, cellular senescence and tissue NAD decline during ageing and offer novel therapeutic opportunities to maintain NAD levels during ageing. Senescent cells in fat and liver are shown to attract M1-like macrophages with increased expression of the NAD-consuming enzyme CD38, leading to their local accumulation and providing a mechanism for the age-associated decline in tissue NAD + levels.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.