ABSTRACT
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.
Subject(s)
Gene Targeting/methods , Macaca fascicularis/genetics , Animals , Base Sequence , Cell Line , Embryo, Mammalian/metabolism , Female , Humans , Molecular Sequence Data , Mosaicism , Sequence AlignmentABSTRACT
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.
Subject(s)
Lysine , Neurons , Animals , Mice , Patch-Clamp Techniques , Pyramidal Cells/physiology , Deubiquitinating Enzyme CYLDABSTRACT
An amendment to this paper has been published and can be accessed via the original article.
ABSTRACT
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.
Subject(s)
Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/pathology , Inflammation/complications , Inflammation/genetics , Liver Neoplasms/etiology , Liver Neoplasms/pathology , Neovascularization, Pathologic/genetics , RNA-Binding Proteins/genetics , Adenosine/analogs & derivatives , Animals , Epigenesis, Genetic , Humans , Methylation , Mice , Models, Biological , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , TranscriptomeABSTRACT
The recent emergence of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has attracted significant attention for its potential to improve traits of agricultural importance. However, most applications in livestock species to date have depended on aberrant DNA repair to generate frameshifting indels. Whether this genomic engineering technique involving homology-dependent repair (HDR) can be used to introduce defined point mutations has been less explored. Previously, we reported a GâA point mutation (g.231A>G, p.Val397Ile) in the growth differentiation factor 9 (GDF9) gene that has a large effect on the litter size of cashmere goats. In the present study we report that by co-injecting synthesised RNAs and single-stranded oligo deoxynucleotide (ssODN) donor sequences into goat zygotes, we successfully introduced defined point mutations resulting in single amino acid substitutions in the proteins as expected. The efficiency of this precise single-nucleotide substitution in newborn kids was as high as 24% (4/17), indicating that ssODN-directed HDR via zygote injection is efficient at introducing point mutations in the goat genome. The findings of the present study further highlight the complex genome modifications facilitated by the CRISPR/Cas9 system, which is able to introduce defined point mutations. This represents a significant development for the improvement of reproduction traits in goats, as well as for validating the roles of specific nucleotides in functional genetic elements in large animals.
Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing/veterinary , Goats/genetics , Growth Differentiation Factor 9/genetics , Litter Size/genetics , Point Mutation , Animals , Animals, Genetically Modified , CRISPR-Associated Proteins/metabolism , Feasibility Studies , Female , Gene Editing/methods , Genotype , Male , PhenotypeABSTRACT
Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/ß-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.
Subject(s)
CRISPR-Associated Proteins/metabolism , Testis/metabolism , Transcription Factors/metabolism , Animals , Animals, Genetically Modified , CRISPR-Associated Proteins/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , DAX-1 Orphan Nuclear Receptor/genetics , DAX-1 Orphan Nuclear Receptor/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Haplorhini , Humans , Hypogonadism/genetics , Hypogonadism/metabolism , Male , Sertoli Cells/metabolism , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Deoxyribonuclease I/metabolism , Endonucleases/metabolism , Genome, Bacterial , Animals , Base Sequence , Deoxyribonuclease I/genetics , Embryo, Mammalian , Gene Expression Regulation, Enzymologic , HeLa Cells , Humans , Mice , Mutation , RNA, BacterialABSTRACT
Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.
Subject(s)
CRISPR-Cas Systems , Gene Deletion , Gene Targeting/methods , Nuclear Proteins/metabolism , Animals , Chromosomes, Mammalian/genetics , Gene Targeting/economics , Genomic Imprinting , Mice , Nuclear Proteins/geneticsABSTRACT
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Transcription Activator-Like Effector Nucleases/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Engineering , HumansABSTRACT
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.
Subject(s)
Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Acute , Rho Guanine Nucleotide Exchange Factors , Humans , Leukemia, Myeloid, Acute/genetics , Prognosis , Research Design , Retrospective Studies , Rho Guanine Nucleotide Exchange Factors/genetics , Signal TransductionABSTRACT
CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Mice , Animals , Humans , CRISPR-Cas Systems/genetics , Adenine/metabolism , Cytosine/metabolism , NucleotidesABSTRACT
A representative closely packed conjugated polyrotaxane (CPR1) is synthesized by threading polyaniline (PAN) into ß-cyclodextrin (CD) macrocycles and utilized for the first time to construct an RRAM device that exhibits an outstanding resistive switching capability. The CPR1 RRAM device displays remarkable nonvolatile memory performance with an extremely high ON/OFF ratio of 108, the ultra-fast response of 29 ns, excellent reliability and reproducibility, and long-term stability (more than 1 year). The mechanism underlying this resistive switching behavior is understood according to the electric-field-induced proton doping of the PAN core by the CD sheath through hydrogen bonding interactions. More impressively, the favorable solubility and intrinsic flexibility of CPR1 allow for large-scale fabrication of flexible CPR1 RRAM device arrays by full-printing technology with endurance of 1000 bending cycles at the minimum bending radius of 3 mm, higher ON/OFF ratio of 108, and relatively lower operating voltage of 1.8 V. This work shows the potential of CPR materials in highly stable memory devices for next-generation flexible and wearable electronics.
ABSTRACT
Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.
Subject(s)
Adenosine Deaminase/genetics , CRISPR-Associated Protein 9/genetics , DNA/genetics , Escherichia coli Proteins/genetics , Gene Editing/methods , Adenine/metabolism , Adenosine Deaminase/metabolism , Bacterial Toxins/genetics , CRISPR-Associated Protein 9/metabolism , Cell Line, Tumor , Cytosine/metabolism , DNA/metabolism , Escherichia coli Proteins/metabolism , Genes, Reporter , HEK293 Cells , Humans , Inosine/genetics , Inosine/metabolism , Protein Engineering , RNA Editing/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA-Seq , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
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
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.
Subject(s)
Antineoplastic Agents/pharmacology , Bile Duct Neoplasms/pathology , Cholangiocarcinoma/pathology , Down-Regulation/drug effects , Early Growth Response Transcription Factors/genetics , Gramicidin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Necrosis/chemically inducedABSTRACT
The ON/OFF ratio and long-term stability are two important issues for flexible organic-inorganic hybrid perovskite (OHP) resistive random access memory (RRAM) for practical applications. In this work, polyvinylammonium (PVAm) is applied to partially replace methylamine ions (MA+) to fabricate the stable and flexible polymeric OHP RRAM devices, wherein PVAm acts as nucleation sites and the template for crystalline growth of MAPbI3 to tune the microscopic perovskite structure. Simultaneously, the multiple perovskite grain interfaces are strengthened through the long-carbochain polymeric backbone, hence producing a continuous and compact perovskite film. As a result, the PVAm-modified OHP RRAM device shows remarkable enhancement of the ON/OFF ratio, long-term stability, and flexibility compared with the unmodified OHP device. Specifically, the polymeric OHP device exhibits fast and stable nonvolatile resistive switching (RS) characteristics with an ON/OFF ratio of â¼105 and a set voltage of -0.45 V under ambient conditions. Also, the distinct multilevel RS behavior can be realized in this device by controlling the compliance current in the SET process. Additionally, the unsealed polymeric OHP device manifests the striking long-term stability, which can still maintain the stable memory performance after 1 year exposure to the humid and thermal ambient environment. Furthermore, the flexible polymeric OHP device was also fabricated and affords the excellent bending endurance behavior by showing a reproducible RS property over 100-cycle bending experiments. This work provides a new perovskite-based material design strategy of polymeric OHP for stable and flexible RRAM devices with the high ON/OFF ratio.
ABSTRACT
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.
Subject(s)
Codon, Nonsense/genetics , Hair/growth & development , Hair/metabolism , Alleles , Animals , Base Pairing/genetics , Blotting, Western , CRISPR-Cas Systems/genetics , Cells, Cultured , Fibroblast Growth Factor 5/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Editing , Goats , Male , Mutation/genetics , Phenotype , Polymorphism, Single Nucleotide/geneticsABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Gene Targeting , Mice, Transgenic , Oocytes/cytology , Alleles , Animals , DNA Primers/genetics , Female , Gene Editing , Genome , Male , Mice , Mice, Inbred C57BL , Mutagenesis , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Polymerase Chain Reaction , RNA, Guide, Kinetoplastida/genetics , Receptors, Androgen/genetics , Sequence Analysis, DNA , Zona Pellucida Glycoproteins/geneticsABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0164640.].
ABSTRACT
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.