The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis.Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloningfree single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F 1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/ Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5 ′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
Heparin is a sulphated polysaccharide, synthesized exclusively by connective-tissue-type mast cells and stored in the secretory granules in complex with histamine and various mast-cell proteases. Although heparin has long been used as an antithrombotic drug, endogenous heparin is not present in the blood, so it cannot have a physiological role in regulating blood coagulation. The biosynthesis of heparin involves a series of enzymatic reactions, including sulphation at various positions. The initial modification step, catalysed by the enzyme glucosaminyl N-deacetylase/N-sulphotransferase-2, NDST-2, is essential for the subsequent reactions. Here we report that mice carrying a targeted disruption of the gene encoding NDST-2 are unable to synthesize sulphated heparin. These NDST-2-deficient mice are viable and fertile but have fewer connective-tissue-type mast cells; these cells have an altered morphology and contain severely reduced amounts of histamine and mast-cell proteases. Our results indicate that one site of physiological action for heparin could be inside connective-tissue-type mast cells, where its absence results in severe defects in the secretory granules.
Zebrafish is a popular model organism for studying development and disease and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including: target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline transmitting mutations by fluorescent PCR; determination of the exact lesion by Sanger or next- generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNA's can be evaluated in 3 days, zebrafish transmitting germline mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.
Heparan sulfate is a sulfated polysaccharide present on most cell surfaces and in the extracellular matrix. In vivo functions of heparan sulfate can be studied in mouse strains lacking enzymes involved in the biosynthesis of heparan sulfate. Glucosaminyl N-deacetylase/ N-sulfotransferase (NDST) catalyzes the first modifying step in the biosynthesis of the polysaccharide. This bifunctional enzyme occurs in several isoforms. We here report that targeted gene disruption of NDST-1 in the mouse results in a structural alteration of heparan sulfate in most basement membranes as revealed by immunohistochemical staining of fetal tissue sections using antibodies raised against heparan sulfate. Biochemical analysis of heparan sulfate purified from fibroblast cultures, lung, and liver of NDST-1-deficient embryos demonstrated a dramatic reduction in N-sulfate content. Most NDST-1-deficient embryos survive until birth; however, they turn out to be cyanotic and die neonatally in a condition resembling respiratory distress syndrome. In addition, a minor proportion of NDST-1-deficient embryos die during the embryonic period. The cause of the embryonic lethality is still obscure, but incompletely penetrant defects of the skull and the eyes have been observed.
During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development.We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/ N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N-and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor  (PDGFR) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.[Keywords: PDGF-B; angiogenesis; heparan sulfate; pericyte; vascular development] Supplemental material is available at http://www.genesdev.org. Tissue morphogenesis depends on cell-cell interactions, controlling directed cell migration proliferation, differentiation, and cell survival. Specificity is often regulated at the level of selective ligand-receptor interaction. However, the spatial distribution and local concentration of the ligand determine the range of the signal, and, as exemplified by morphogens of the hedgehog, TGF, and Wnt family members, also the nature of the signal. Indeed, spatial restriction defines the activities of most peptide growth factors and many secreted neural guidance molecules. In vascular development, peptide growth factors of the VEGF and platelet-derived growth factor (PDGF) families regulate the migration and proliferation of endothelial cells and supporting mural cells; i.e., pericytes (PC) and vascular smooth muscle cells (vSMC). The longitudinal migration and proliferation of vSMC/PC depend on paracrine signaling of endothelial derived PDGF-B to PDGF receptor- (PDGFR) expressed on vSMC/PC (Lindahl et al. 1997;Hellström et al. 1999). PDGF-B is secreted as a homodimer (PDGF-BB), which signals by mediating dimerization of its receptor. Conditional inactivation of Pdgf-b in the endothelium demonstrated that endothelial cells are the
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