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The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing. [ABSTRACT FROM AUTHOR]

The bacterial clustered regularly interspaced short palindromic repeats ( CRISPR)/ CRISPR-associated 9 (Cas9) system is a versatile RNA-guided mammalian genome modification system. One-step generation of mouse genome targeting has been achieved by co-microinjection of one-cell stage embryos with Cas9 m RNA and small/single guide (sg) RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sg RNAs to target an individual gene significantly improved the Cas9-mediated genome targeting with a bi-allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site-dependent off-target effects, and also that the apolipoprotein E gene knockout-mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9-mediated heritable gene modification. Our results provide a dual sg RNAs strategy to facilitate CRISPR/Cas9-mediated mouse genome targeting. [ABSTRACT FROM AUTHOR]

Highlights: [•] Embryo co-microinjection of Cas9 mRNA and multiple sgRNAs leads to heritable multiple gene modifications. [•] One-step generating multiple immunodeficient mouse models with multiallelic mutations. [•] Optimal concentration of Cas9 mRNA and sgRNAs improved targeting efficiency. [•] Targeting one site with two adjacent sgRNAs increases Cas9:sgRNA-mediated targeting efficiency and bi-allelic mutations. [ABSTRACT FROM AUTHOR]

Biocytin, a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine, has been used as a histological dye to stain nerve cells. Electrophysiological activity and morphology are two key characteristics of neurons, but revealing both the electrophysiological and morphological properties of the same neuron is challenging. This article introduces a detailed and easy-to-operate procedure for single-cell labeling in combination with whole-cell patch-clamp recording. Using a recording electrode filled with a biocytin-containing internal solution, we demonstrate the electrophysiological and morphological characteristics of pyramidal (PNs), medial spiny (MSNs) and parvalbumin neurons (PVs) in brain slices, where the electrophysiological and morphological properties of the same individual cell are elucidated. We first introduce a protocol for whole-cell patch-clamp recording in various neurons, coupled with the intracellular diffusion of biocytin delivered by the glass capillary of the recording electrode, followed by a post hoc procedure to reveal the architecture and morphology of biocytin-labeled neurons. An analysis of action potentials (APs) and neuronal morphology, including the dendritic length, number of intersections, and spine density of biocytin-labeled neurons, were performed using ClampFit and Fiji Image (ImageJ), respectively. Next, to take advantage of the techniques introduced above, we uncovered defects in the APs and the dendritic spines of PNs in the primary motor cortex (M1) of deubiquitinase cylindromatosis (CYLD) knock-out (Cyld−/−) mice. In summary, this article provides a detailed methodology for revealing the morphology as well as the electrophysiological activity of a single neuron that will have many applications in neurobiology. [ABSTRACT FROM AUTHOR]

The roles of Rho GTPases in various types of cancer have been extensively studied, but the research of Rho guanine nucleotide exchange factors (GEFs) in cancer is not comprehensive. Rho guanine nucleotide exchange factor 6 (ARHGEF6) is an important member of the Rho GEFs family involved in cytoskeletal rearrangement, and it has not been investigated in acute myeloid leukemia (AML). Our research showed that the expression of ARHGEF6 was mainly higher in AML cell lines, meanwhile, was highest in the samples from patients with AML compared to other cancer types. High ARHGEF6 expression in AML was associated with a good prognosis. ARHGEF6low cases showed significantly higher overall survival (OS) after autologous or allogeneic HSCT (auto/allo-HSCT). High expression of ARHGEF6 downregulates the negative regulation of myeloid differentiation process and upregulates G protein-coupled receptor signaling pathway-related processes, among which HOXA9, HOXB6, and TRH have significant differential expression and prognostic impact in AML. Therefore, ARHGEF6 can become a prognostic marker in AML; ARHGEF6low patients can gain from auto/allo-HSCT. [ABSTRACT FROM AUTHOR]

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes. [ABSTRACT FROM AUTHOR]

Gramicidin is a well-known antibiotic and recently was reported to induced tumour cell death, however, little is understood about the molecular mechanism of gramicidin as a therapeutic agent for solid tumours. Here, we investigated the role of gramicidin in cholangiocarcinoma cells. We found that gramicidin A inhibits cholangiocarcinoma cell growth and induced the necrotic cell death. We used next generation sequencing to analyse gene expression profiles of cholangiocarcinoma cells treated with gramicidin. We identified 265 differentially expressed genes in cholangiocarcinoma cells between PBS treatment and gramicidin treatment. EGR4 was confirmed to be a target of gramicidin-induced cell growth inhibition. Furthermore, we demonstrated that downregulation of EGR4 in cholangiocarcinoma cells leads to restraining tumour cell growth. Of note, EGR4 was expressed at highest levels in cholangiocarcinoma tissues among 17 types of human cancers, and EGR4 expression positively correlated with several growth factors associated with cholangiocarcinoma. Our findings ascertain that EGR4 is a potential target in cholangiocarcinoma and suppressing EGR4 by gramicidin establish an essential mechanism for bile duct carcinoma progression. [ABSTRACT FROM AUTHOR]

Recyclable low-temperature phase change microcapsules (LTPCMs) have the potential applications in the short-distance cold chain transportation due to their reliable reusability in cold storage. Herein, LTPCMs are synthesized via in-situ suspension copolymerization of styrene and methyl methacrylate in absence of harm substances, providing the non-crosslinking copolymer shells. n -Dodecane, n -tridecane and n -tetradecane, inducing the microphase separation of non-crosslinking copolymers, are successfully encapsulated to achieve n -do-LTPCMs, n -tri-LTPCMs and n -tetra-LTPCMs, which respectively bear the high phase change enthalpy of 110.53 J·g−1 at −8.69 °C, 38.33 J·g−1/93.71 J·g−1 at −17.61 °C/−4.96 °C and 166.79 J·g−1 at 8.59 °C and subsequently show the cold-discharging periods of 30 min, 40 min and 120 min. The multiple circulation of cold-discharging process indicates the excellent recyclability for cold storage owing to their unchanged cold-discharging period. Especially, n -tetra-LTPCM-65 bears the best comprehensive cold-storing performance in all the previously reported LTPCMs, such as narrow cold-discharging temperature range of 3–4 °C, long cold-discharging period of 69–120 min and low cold-discharging capacity of 33.4 J·g−1·K−1. This work successfully provided the recyclable LTPCMs for cold storage in the short-distance cold chain transportation. [ABSTRACT FROM AUTHOR]

The ability to alter single bases without homology directed repair (HDR) of double‐strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C‐to‐T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single‐cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole‐genome resequencing analyses for off‐target changes caused by BE3 were low at a genome‐wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single‐cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation. [ABSTRACT FROM AUTHOR]

Background: Dynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m6A-methylation in hepatocellular carcinoma (HCC). Results: Using liquid chromatography-tandem mass spectrometry and m6A-immunoprecipitation in combination with high-throughput sequencing, we determined the m6A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m6A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m6A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer. Conclusion: Our results have characterized the m6A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular 'rheostat' in epitranscriptome and cancer progression. [ABSTRACT FROM AUTHOR]

Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations. [ABSTRACT FROM AUTHOR]

Summary: Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding. [ABSTRACT FROM AUTHOR]

Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs. [ABSTRACT FROM AUTHOR]