Grass carp peroxiredoxin 5 and 6-mediated autophagy inhibit grass carp reovirus replication and mitigate oxidative stress.

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H2O2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H2O2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress.

Grass carp superoxide dismutases exert antioxidant function and inhibit autophagy to promote grass carp reovirus (GCRV) replication.

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two CuZn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H2O2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication.

Production of Astaxanthin by Animal Cells via Introduction of an Entire Astaxanthin Biosynthetic Pathway.

Astaxanthin is a fascinating molecule with powerful antioxidant activity, synthesized exclusively by specific microorganisms and higher plants. To expand astaxanthin production, numerous studies have employed metabolic engineering to introduce and optimize astaxanthin biosynthetic pathways in microorganisms and plant hosts. Here, we report the metabolic engineering of animal cells in vitro to biosynthesize astaxanthin. This was accomplished through a two-step study to introduce the entire astaxanthin pathway into human embryonic kidney cells (HEK293T). First, we introduced the astaxanthin biosynthesis sub-pathway (Ast subp) using several genes encoding ß-carotene ketolase and ß-carotene hydroxylase enzymes to synthesize astaxanthin directly from ß-carotene. Next, we introduced a ß-carotene biosynthesis sub-pathway (ß-Car subp) with selected genes involved in Ast subp to synthesize astaxanthin from geranylgeranyl diphosphate (GGPP). As a result, we unprecedentedly enabled HEK293T cells to biosynthesize free astaxanthin from GGPP with a concentration of 41.86 µg/g dry weight (DW), which represented 66.19% of the total ketocarotenoids (63.24 µg/g DW). Through optimization steps using critical factors in the astaxanthin biosynthetic process, a remarkable 4.14-fold increase in total ketocarotenoids (262.10 µg/g DW) was achieved, with astaxanthin constituting over 88.82%. This pioneering study holds significant implications for transgenic animals, potentially revolutionizing the global demand for astaxanthin, particularly within the aquaculture sector.

Single-cell analysis reveals an Angpt4-initiated EPDC-EC-CM cellular coordination cascade during heart regeneration.

Mammals exhibit limited heart regeneration ability, which can lead to heart failure after myocardial infarction. In contrast, zebrafish exhibit remarkable cardiac regeneration capacity. Several cell types and signaling pathways have been reported to participate in this process. However, a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable. We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration. We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes, and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration. Furthermore, we identified a regeneration-induced cell (RIC) population in the epicardium-derived cells (EPDC), and demonstrated Angiopoietin 4 (Angpt4) as a specific regulator of heart regeneration. angpt4 expression is specifically and transiently activated in RIC, which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway, and further induces activation of cathepsin K in cardiomyocytes through RA signaling. Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation, while overexpression of angpt4 accelerates regeneration. Furthermore, we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes, and promote cardiac repair in mice after myocardial infarction, indicating that the function of Angpt4 is conserved in mammals. Our study provides a mechanistic understanding of heart regeneration at single-cell precision, identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration, and offers a novel therapeutic target for improved recovery after human heart injuries.

Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set.

Image-based identification and quantification of different types of spermatogenic cells is of great importance, not only for reproductive studies but also for genetic breeding. Here, we have developed antibodies against spermatogenesis-related proteins in zebrafish (Danio rerio), including Ddx4, Piwil1, Sycp3, and Pcna, and a high-throughput method for immunofluorescence analysis of zebrafish testicular sections. By immunofluorescence analysis of zebrafish testes, our results demonstrate that the expression of Ddx4 decreases progressively during spermatogenesis, Piwil1 is strongly expressed in type A spermatogonia and moderately expressed in type B spermatogonia, and Sycp3 has distinct expression patterns in different subtypes of spermatocytes. Additionally, we observed polar expression of Sycp3 and Pcna in primary spermatocytes at the leptotene stage. By a triple staining of Ddx4, Sycp3, and Pcna, different types/subtypes of spermatogenic cells were easily characterized. We further demonstrated the practicality of our antibodies in other fish species, including Chinese rare minnow (Gobiocypris rarus), common carp (Cyprinus carpio), blunt snout bream (Megalobrama amblycephala), rice field eel (Monopterus albus) and grass carp (Ctenopharyngodon idella). Finally, we proposed an integrated criterion for identifying different types/subtypes of spermatogenic cells in zebrafish and other fishes using this high-throughput immunofluorescence approach based on these antibodies. Therefore, our study provides a simple, practical, and efficient tool for the study of spermatogenesis in fish species.

miR-2188-5p promotes GCRV replication by the targeted degradation of klf2a in Ctenopharyngodon idellus.

Studies on host immunity evasion by aquatic viruses have largely focused on coding genes. There is accumulating evidence for the important biological functions of non-coding miRNAs in virus-host interactions. The regulatory functions of non-coding miRNAs in fish reovirus-host interactions remain unknown. Here, miR-2188-5p in grass carp (Ctenopharyngodon idellus), a miRNA specific to teleosts, was predicted to target the 3' UTR of the transcription factor klf2a. A correlation analysis and dual-luciferase reporter assay revealed that miR-2188-5p could induce the degradation of klf2a. The expression of miR-2188-5p induced the degradation of klf2a in a dose-dependent manner, suppressing the type I interferon response and promoting grass carp reovirus (GCRV) replication. As determined by a co-expression analysis, klf2a inhibited viral infection when miR-2188-5p was overexpressed. The targeted degradation of klf2a by miR-2188-5p could inhibit the type I interferon response and promote the replication of GCRV; however, this targeted degradation ability was insufficient to fully inhibit GCRV infection. These results provide novel insights into the regulatory effects and biological functions of non-coding miRNAs in fish-virus interactions.

Overexpression of growth hormone improved hepatic glucose catabolism and relieved liver lipid deposition in common carp (Cyprinus carpio L.) fed a high-starch diet.

Growth hormone (GH) is important for regulating insulin secretion and carbohydrate metabolism, and its role in mammalian models of diabetes is relatively worked out. Although some fish species were used as models for diabetes research, the effects of GH on insulin and glucose catabolism and anabolism in these models remain to be clarified. In this study, we investigated the effect of GH on insulin and glucose catabolism and anabolism in an omnivorous fish using GH transgenic (T) common carp that consistently overexpressed GH and wild-type (WT) common carp. We compared the intestinal morphology, and digestive and absorptive capacity of fish fed commercial feed. We also analyzed the growth performance, insulin level, glucose catabolism and anabolism, lipid deposition, and lipid catabolism and anabolism in T carp and WT carp fed diets containing either 30% or 40% starch. In the intestine of T carp, α-amylase activity was enhanced, the number of goblet cells and intestinal villi surface area was increased, and the expression level of glucose transport protein-related genes (glut2 and sglt1) was upregulated when compared to these indicators in WT carp. When fed either a normal or high-starch diet, the growth performance of T carp was better than that of WT carp. Compared with WT carp, serum insulin was increased and glucose was decreased, hepatic expression level of igf-1 and glycolysis-related genes was increased, and the activity level of a hepatic enzyme related to glycolysis was enhanced in T carp. When fed with a high-starch diet, the serum alanine aminotransferase activity, hepatic lipid content, and malondialdehyde content were significantly lower in T carp than in WT carp. These results indicated that overexpression of GH (1) enhanced carbohydrate digestion and absorption in the carp intestine, (2) did not induce insulin resistance and improved glucose catabolism and utilization in carp, and (3) relieved liver lipid deposition. Our data might provide new insights into potential ways to improve glucose utilization in fish and diabetes treatments.

Krüppel-like factor 2a (KLF2A) suppresses GCRV replication by upregulating serpinc1 expression in Ctenopharyngodon idellus kidney (CIK) cells.

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2, serpinc1, serpinh1a, and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish.

Grass Carp Reovirus Induces Formation of Lipid Droplets as Sites for Its Replication and Assembly.

Grass carp is an important commercial fish in China that is plagued by various diseases, especially the hemorrhagic disease induced by grass carp reovirus (GCRV). Nevertheless, the mechanism by which GCRV hijacks the host metabolism to complete its life cycle is unclear. In this study, we performed lipidomic analysis of grass carp liver samples collected before and after GCRV infection. GCRV infection altered host lipid metabolism and increased de novo fatty acid synthesis. Increased de novo fatty acid synthesis induced accumulation of lipid droplets (LDs). LDs are associated with GCRV viroplasms, as well as viral proteins and double-stranded RNA. Pharmacological inhibition of LD formation led to the disappearance of viroplasms, accompanied by decreased viral replication capacity. Moreover, transmission electron microscopy revealed LDs in close association with the viroplasms and mounted GCRV particles. Collectively, these data suggest that LDs are essential for viroplasm formation and are sites for GCRV replication and assembly. Our results revealed the detailed molecular events of GCRV hijacking host lipid metabolism to benefit its replication and assembly, which may provide new perspective for the prevention and control of GCRV. IMPORTANCE Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, which belongs to the family Reoviridae. GCRV-induced hemorrhagic disease is a major threat to the grass carp aquaculture industry. Viruses are obligate intracellular parasites that require host cell machinery to complete their life cycle; the mechanism by which GCRV hijacks the host metabolism to benefit viral replication and assembly remains unclear. Our study demonstrated that GCRV infection alters host lipid metabolism and increases de novo fatty acid synthesis. The increased de novo fatty acid synthesis induced accumulation of LDs, which act as sites or scaffolds for GCRV replication and assembly. Our findings illustrate a typical example of how the virus hijacks cellular organelles for replication and assembly and hence may provide new insights for the prevention and control of GCRV.

Grass Carp Prx 3 Elevates Host Antioxidant Activity and Induces Autophagy to Inhibit Grass Carp Reovirus (GCRV) Replication.

Peroxiredoxins are a family of antioxidant proteins that protect cells from oxidative damage caused by reactive oxygen species (ROS). Herein, the peroxiredoxin 3 gene from grass carp (Ctenopharyngodon idellus), named CiPrx3, was cloned and analyzed. The full-length cDNA of CiPrx3 is 1068 bp long, with a 753 bp open reading frame (ORF) that contains a thioredoxin-2 domain, two peroxiredoxin signature motifs, and two highly conserved cysteine residues. CiPrx3 was ubiquitously expressed in all the tested tissues, while its expression level was altered significantly after exposure to grass carp reovirus (GCRV) and pathogen-associated molecular patterns (PAMPs). CiPrx3 was localized in the mitochondria of transfected cells and concentrated in the nucleus after poly (I:C) treatment. Transformation of CiPrx3 into Escherichia coli enhanced host resistance to H2O2 and heavy metals. Purified recombinant CiPrx3 proteins could protect DNA against oxidative damage. Overexpression of CiPrx3 in fish cells reduced intracellular ROS, increased cell viability, and decreased cell apoptosis caused by H2O2 stimulation and GCRV infection. Further study indicated that CiPrx3 induced autophagy to inhibit GCRV replication in fish cells. Collectively, these results imply that grass carp Prx3 elevates host antioxidant activity and induces autophagy to inhibit GCRV replication.

Grass carp SERPINA1 inhibits GCRV infection through degrading CF2.

SERPINA1, a member of the serine protease inhibitor family, plays a role in viral infection and inflammation by regulating the activities of serine and cysteine proteases. To date, there have been no reports on the immune function of SERPINA1 in fishes. In this study, we first cloned the serpina1 gene of grass carp (Ctenopharyngodon idellus) and found that it could respond rapidly to the infection of Grass carp reovirus (GCRV), and overexpression of serpina1 could enhance the antiviral response of CIK cells. A polyclonal antibody of SERPINA1 was prepared, and the protein interacting with SERPINA1 was screened by CoIP/MS in grass carp hepatopancreas tissue. It was found that SERPINA1 interacted with coagulation factor 2 (CF2) and could degrade it in a dose-dependent manner. In addition, overexpression of cf2 contributed to the infection of GCRV in CIK cells, whereas co-expression of serpina1 and cf2 in grass carp reduced the copy number of GCRV in cells. The results showed that grass carp SERPINA1 could inhibit GCRV infection by degrading CF2. This study proposes that SERPINA1 can inhibit viral infection through interaction with the coagulation factor, providing new insights into the molecular mechanism of SERPINA1's antiviral function.

Improved liver lipid catabolism and utilization in growth hormone transgenic common carp (Cyprinus carpio L.) through enhanced lipolytic and fatty acid ß-oxidation pathways.

Growth hormone (GH) transgenic common carp (Cyprinus carpio L.) show desirable aquaculture traits. Their specific growth rate (SGR) and feed efficiency (FE) are approximately 12% and 17% higher than the wild-type (WT) common carp, respectively. However, the mechanisms of lipid catabolism (lipolysis and fatty acid ß-oxidation) and utilization in GH transgenic common carp are still unclear. In this study, we firstly compared the lipid metabolism of GH transgenic (initial weight 3.72 ± 0.32 g) and WT (initial weight 3.30 ± 0.28 g) common carp fed with a normal fat level diet (6% lipid, 33% protein) for two months, then compared the growth performance of GH transgenic (initial weight 3.65 ± 0.33 g) and WT (initial weight 3.27 ± 0.26 g) common carp fed with different fat levels diets (6% lipid and 12% lipid, 33% protein) for two months. We found that the lipid content in serum, liver and whole body was significantly reduced in GH transgenic common carp, the hepatic activities of the lipolytic enzymes hormone-sensitive lipase and adipose triglyceride lipase were enhanced, and the hepatic expression level of hormone-sensitive lipase was upregulated. In addition, the mitochondrion numbers were increased, and the expression level of carnitine palmitoyltransferase-1a and carnitine palmitoyltransferase-1b was upregulated in the liver of GH transgenic common carp. GH transgenic common carp showed higher weight gain and SGR than that in WT carp when fed with a normal-fat diet as they did when fed with a high-fat diet, and GH transgenic common carp showed higher FE than that in WT carp when fed with a high-fat diet. These results suggested that the lipid catabolism and utilization was improved in the GH transgenic common carp liver through enhanced lipolytic and fatty acid ß-oxidation pathways. Our study provides new insights into improving lipid utilization in some aquaculture fish species.

Isolation, Identification, and Investigation of Pathogenic Bacteria From Common Carp (Cyprinus carpio) Naturally Infected With Plesiomonas shigelloides.

Various bacterial diseases have caused great economic losses to the high-density and intensive aquaculture industry; however, the pathogenic mechanism underlying the large-scale challenged to caused by many bacteria remain unclear, making the prevention and treatment of these diseases difficult. In the present study, we isolated a bacterial strain from Cyprinus carpio having a typical bacterial disease and named it Cc2021. Through subsequent morphological observations, a regression challenge, biochemical identification, and 16S rRNA gene sequence analysis, we determined Cc2021 to be Plesiomonas shigelloides. Subsequently, we comprehensively investigated the pathogenicity of P. shigelloides in C. carpio through a regression challenge and assessed the underlying the pathogenic mechanism. Mortality results revealed that P. shigelloides is highly pathogenic and infects various tissues throughout the body, resulting in edema of the liver, spleen, and body and head kidneys. Histopathological analysis revealed obvious inflammation, bleeding, and necrosis in the intestine, spleen, and head kidney. The body's immune tissues actively produce complement C3, superoxide dismutase, and lysozyme after a challenge to resist bacterial invasion. With regard to the underlying pathogenesis of P. shigelloides, comparative transcriptome analysis revealed 876 upregulated genes and 828 downregulated genes in the intestine of C. carpio after the challenge. Analysis of differentially expressed unigenes revealed the involvement of major immune pathways, particularly the TNF signaling pathway, interleukin (IL)-17 signaling pathway, and Toll-like receptor signaling pathway. The present study provides new valuable information on the immune system and defense mechanisms of P. shigelloides.

Screening of Genes Related to Sex Determination and Differentiation in Mandarin Fish (Siniperca chuatsi).

Mandarin fish has an XX/XY sex-determination system. The female mandarin fish is typically larger than the male. Sex identification and the discovery of genes related to sex determination in mandarin fish have important theoretical significance in the elucidation of the regulation and evolutionary mechanism of animal reproductive development. In this study, the chromosome-level genome of a female mandarin fish was assembled, and we found that LG24 of the genome was an X chromosome. A total of 61 genes on the X chromosome showed sex-biased expression. Only six gonadal genes (LG24G00426, LG24G003280, LG24G003300, LG24G003730, LG24G004200, and LG24G004770) were expressed in the testes, and the expression of the other gene LG24G003870 isoform 1 in the ovaries was significantly higher than that in the testes (p < 0.01). Five (except LG24G003280 and LG24G003300) of the seven aforementioned genes were expressed at the embryonic development stage, suggesting their involvement in early sex determination. The expression of LG24G004770 (encoding HS6ST 3-B-like) was also significantly higher in female muscles than in male muscles (p < 0.01), indicating other functions related to female growth. ZP3 encoded by LG24G003870 isoform 1 increased the C-terminal transmembrane domain, compared with that encoded by other fish zp3 isoforms, indicating their different functions in sex determination or differentiation. This study provides a foundation for the identification of sex-determining genes in mandarin fish.

Genome-wide DNA methylation reveals potential epigenetic mechanism of age-dependent viral susceptibility in grass carp.

BACKGROUND: Grass carp are an important farmed fish in China that are infected by many pathogens, especially grass carp reovirus (GCRV). Notably, grass carp showed age-dependent susceptibility to GCRV; that is, grass carp not older than one year were sensitive to GCRV, while those over three years old were resistant to this virus. However, the underlying mechanism remains unclear. Herein, whole genome-wide DNA methylation and gene expression variations between susceptible five-month-old (FMO) and resistant three-year-old (TYO) grass carp were investigated aiming to uncover potential epigenetic mechanisms. RESULTS: Colorimetric quantification revealed that the global methylation level in TYO fish was higher than that in FMO fish. Whole-genome bisulfite sequencing (WGBS) of the two groups revealed 6214 differentially methylated regions (DMRs) and 4052 differentially methylated genes (DMGs), with most DMRs and DMGs showing hypermethylation patterns in TYO fish. Correlation analysis revealed that DNA hypomethylation in promoter regions and DNA hypermethylation in gene body regions were associated with gene expression. Enrichment analysis revealed that promoter hypo-DMGs in TYO fish were significantly enriched in typical immune response pathways, whereas gene body hyper-DMGs in TYO fish were significantly enriched in terms related to RNA transcription, biosynthesis, and energy production. RNA-seq analysis of the corresponding samples indicated that most of the genes in the above terms were upregulated in TYO fish. Moreover, gene function analysis revealed that the two genes involved in energy metabolism displayed antiviral effects. CONCLUSIONS: Collectively, these results revealed genome-wide variations in DNA methylation between grass carp of different ages. DNA methylation and gene expression variations in genes involved in immune response, biosynthesis, and energy production may contribute to age-dependent susceptibility to GCRV in grass carp. Our results provide important information for disease-resistant breeding programs for grass carp and may also benefit research on age-dependent diseases in humans.

Sinhcaf-dependent histone deacetylation is essential for primordial germ cell specification.

Primordial germ cells (PGCs) are the progenitor cells that give rise to sperm and eggs. Sinhcaf is a recently identified subunit of the Sin3 histone deacetylase complex (SIN3A-HDAC). Here, we provide evidence that Sinhcaf-dependent histone deacetylation is essential for germ plasm aggregation and primordial germ cell specification. Specifically, maternal-zygotic sinhcaf zebrafish mutants exhibit germ plasm aggregation defects, decreased PGC abundance and male-biased sex ratio, which can be rescued by re-expressing sinhcaf. Overexpression of sinhcaf results in excess PGCs and a female-biased sex ratio. Sinhcaf binds to the promoter region of kif26ab. Loss of sinhcaf epigenetically switches off kif26ab expression by increasing histone 3 acetylation in the promoter region. Injection of kif26ab mRNA could partially rescue the germ plasm aggregation defects in sinhcaf mutant embryos. Taken together, we demonstrate a role of Sinhcaf in germ plasm aggregation and PGC specialization that is mediated by regulating the histone acetylation status of the kif26ab promoter to activate its transcription. Our findings provide novel insights into the function and regulatory mechanisms of Sinhcaf-mediated histone deacetylation in PGC specification.

Complete Depletion of Primordial Germ Cells Results in Masculinization of Monopterus albus, a Protogynous Hermaphroditic Fish.

Primordial germ cells (PGCs) play an important role in sexual fate determination and gonadal development in gonochoristic fish, such as zebrafish and medaka. However, little is known about the function of PGCs in hermaphroditic fish. Rice field eel (Monopterus albus), a protogynous hermaphroditic fish, is an economically valuable aquaculture species. We eliminated PGCs in rice field eels during embryogenesis via morpholino-mediated knockdown dead end (dnd). The PGCs-depleted gonads developed into testis-like structures with Sertoli cells and Leydig cells. The gene expression pattern of 15-month-old PGCs-depleted gonads showed that male-biased genes, dmrt1, sox9a, gsdf, and amh, were significantly higher than that of the WT, whereas female-biased genes, foxl2 and cyp19a1a, were significantly decreased. These results indicate that PGCs are essential for ovarian differentiation in rice field eel, and PGCs-depleted gonads develop into sterile males without undergoing the female and intersex stages. Our study is the first to identify the role of PGCs in sex differentiation in rice field eel, a protogynous hermaphrodite teleost. And it is of great significance in rice field eel for discovering the underlying mechanism of sex differentiation and establishing sex control technology.

ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish.

In zebrafish, RNA-guided endonucleases such as Cas9 have enabled straightforward gene knockout and the construction of reporter lines or conditional alleles via targeted knockin strategies. However, the performance of another commonly used CRISPR system, Cas12a, is significantly limited due to both the requirement of delivery as purified protein and the necessity of heatshock of injected embryos. To explore the potential of CRISPR/Cas12a-mediated genome editing and simplify its application in zebrafish, we took advantage of the recently reported mRNA-active ErCas12a and investigated its efficacy for the knockin of large DNA fragments, such as fluorescent reporter genes. For knockin via either microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, ErCas12a-injected embryos with a brief heatshock displayed comparable knockin efficiency with Cas9 injection. Through the fusion of T5 exonuclease (T5exo) to the N-terminus of ErCas12a (T5exo-ErCas12a), we further demonstrated high efficiency gene knockout and knockin at a normal incubation temperature, eliminating the embryo-damaging heatshock step. In summary, our results demonstrate the feasibility of ErCas12a- and T5exo-ErCas12a-mediated genome manipulation under simplified conditions, and further expand the genome editing toolbox for various applications in zebrafish.

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation.

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

De novo screening of disease-resistant genes from the chromosome-level genome of rare minnow using CRISPR-cas9 random mutation.

BACKGROUND: Mutants are important for the discovery of functional genes and creation of germplasm resources. Mutant acquisition depends on the efficiency of mutation technology and screening methods. CRISPR-Cas9 technology is an efficient gene editing technology mainly used for editing a few genes or target sites, which has not been applied for the construction of random mutant libraries and for the de novo discovery of functional genes. RESULTS: In this study, we first sequenced and assembled the chromosome-level genome of wild-type rare minnow (Gobiocypris rarus) as a susceptible model of hemorrhagic disease, obtained a 956.05 Mb genome sequence, assembled the sequence into 25 chromosomes, and annotated 26,861 protein-coding genes. Thereafter, CRISPR-Cas9 technology was applied to randomly mutate the whole genome of rare minnow with the conserved bases (TATAWAW and ATG) of the promoter and coding regions as the target sites. The survival rate of hemorrhagic disease in the rare minnow gradually increased from 0% (the entire wild-type population died after infection) to 38.24% (F3 generation). Finally, 7 susceptible genes were identified via genome comparative analysis and cell-level verification based on the rare minnow genome. CONCLUSIONS: The results provided the genomic resources for wild-type rare minnow, and confirmed that the random mutation system designed using CRISPR-Cas9 technology in this study is simple and efficient and is suitable for the de novo discovery of functional genes and creation of a germplasm resource related to qualitative traits.