AQUACULTURE Volume 565, 25 February 2023, 739130 https://doi.org/10.1016/j.aquaculture.2022.739130Get rights and content HIGHLIGHTS * • Multi-locus gene editing was performed in a

hermaphroditic fish. * • _cyp19a1a_ and _foxl2_ was effectively knock out in _Monopterus albus_. * • _cyp19a1a_ plays a key role in regulating ovarian development in _Monopterus albus_.

ABSTRACT Homozygous mutant populations are typically obtained when studying gene function using gene editing. However, because of asynchronous testicular and ovarian development, it is

difficult to obtain homozygous mutant offspring of hermaphrodite fish by mating males and females. The rice field eel (_Monopterus albus_) was the first hermaphrodite teleost to be

targeted seven and 11 sites in the coding regions of these genes, respectively. Fertilized eggs were co-injected with the Cas9 protein and the guide RNAs of each gene. The efficiency of gene

editing–induced mutations in _cyp19a1a_ and _foxl2_ was approximately 100% in the microinjected embryos and adult fish tissues. In _cyp19a1a_ mutants, serum E2 levels were decreased, and

ovarian development was blocked. Only germ stem cells or oogonia were present in the gonads of the 12-month-old _cyp19a1a_ mutant fish. These results suggest that _cyp19a1a_ plays a key role

in regulating primary gonadal development in the ovaries of rice field eels. In _foxl2_ mutants, _cyp19a1a_ transcription and serum E2 levels were minimally decreased, whereas the

transcription of _foxl2-l_, _foxl3_, and _dmrt1_ was increased. Nevertheless, the gonads of _foxl2_ mutant fish differentiated into ovaries, thereby indicating that _foxl2_ does not directly

affect ovarian development in rice field eels. The multi-locus gene editing method established in this study provides an effective approach for studying gene function study and improving

the genetic traits in rice field eels. INTRODUCTION The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system possesses two crucial

components, the guide RNA (gRNA) and the Cas9 protein, which enable effective genome editing in eukaryotic cells. Under the guidance of gRNA, Cas9 specifically targets a genomic locus via

base pairing and induces DNA double-strand breaks (Jinek et al., 2012; Mali et al., 2013). Thereafter, cells use either non-homologous end-joining (NHEJ) or homology-directed repair (HDR)

mechanisms to repair the damage (Hefferin and Tomkinson, 2005; Dai et al., 2010). Generally, the NHEJ pathway results in insertions or deletions (indels) at cleavage sites. The CRISPR/Cas9

system has been widely used in studying gene function and for genetic improvement in teleosts, such as zebrafish (_Danio rerio_) (Hwang et al., 2013) and medaka (_Oryzias latipes_) (Fang et

al., 2018), and commercial fish, such as grass carp (_Ctenopharyngodon idella_) (Ma et al., 2018), sterlet (_Acipenser ruthenus_) (Chen et al., 2018), sea lamprey (_Petromyzon marinus_)

(Square et al., 2015), and loach (_Misgurnus anguillicaudatus_) (Tao et al., 2021). P0 individuals altered using the CRISPR/Cas9 system are usually chimeras, making it difficult to generate

strong and consistent phenotypes (Yen et al., 2014). Furthermore, crossing successive P0 individuals with wild-type fish and self-crossing heterozygous fish to obtain homozygous mutations is

laborious, particularly for hermaphroditic fish. Therefore, improvements in gene editing techniques are needed to achieve high knockout efficiency. Previous studies have shown that

increasing the quantity of injected Cas9 protein improves the gene mutation efficiency, leading to an increase in the mutation rate of the _myostatin_ gene by up to 74% in the P0 generation

of loach (Tao et al., 2021). An in vitro assessment of sgRNA cutting activity has been used to screen for highly effective sgRNA target sites, which improved mutant efficiency in F0

anemonefish (_Amphiprion ocellaris_) embryos by 75–100% (Mitchell et al., 2021). Nonetheless, these modifications do not result in complete knockout. The rice field eel (_Monopterus albus_)

is a protogynous, hermaphroditic teleost. During the initial period of sexual maturation, females sexually revert to males after spawning (Zhou and Gui, 2016). Sex reversal in hermaphroditic

fish is a process of gonadal redifferentiation. Thus, the rice field eel is an ideal model for studying the mechanisms of vertebrate sex differentiation. Moreover, its rapid growth rate and

excellent flesh quality make it an economically important aquaculture species in East and Southeast Asia (Yang et al., 2018). However, fish fry production continues to depend on wild

catches. Hence, the aquaculture industry necessitates the development of breeding technology and breeding varieties with desirable traits, such as controllable sex and rapid growth.

Approximately 30,000 species of teleosts, the largest group of vertebrates, have been discovered, 2% of which are hermaphroditic, including protogynous, protandrous, and bidirectional

sex-changing species (Avise et al., 2009; Gemmell et al., 2019). Gene editing has been successfully performed in a few hermaphroditic fish, such as anemonefish (Mitchel et al., 2021) and

rice field eels (Feng et al., 2017; Luo et al., 2019). However, no homozygous mutant has been obtained becausematuration of ovaries and testes is not synchronized in heterozygous population,

and homozygous individuals cannot be obtained via hermaphrodite mating. Because of the lack of an efficient gene knockout method, the functions of genes that regulate sex differentiation

and sex change in rice field eels remain unclear. Estrogen is a key regulator of ovarian differentiation in teleosts. Aromatase, the rate-limiting enzyme in the estrogen synthesis pathway,

catalyzes the conversion of androgens (testosterone and androstenedione) into estrogens (17β-estradiol and estrone). Two homologous genes encoding aromatase, _cyp19a1a_ and _cyp19a1b_, have

been identified in fish; _cyp19a1b_ is expressed in the brain, whereas _cyp19a1a_ is primarily expressed in the gonads (Chang et al., 2005), which plays an important role in ovarian

differentiation and maintenance (Guiguen et al., 2010; Yin et al., 2017). Our previous study showed that oral administration of letrozole, an aromatase inhibitor, induced juvenile rice field

eels to develop into males (Jiang et al., 2022). Foxl2, a member of the forkhead transcription factor family, is primarily expressed in follicular layer cells of the ovary. In zebrafish,

the disruption of one of the _foxl2_ homologs, _foxl2a_ or _foxl2b_, results in premature ovarian failure or partial sex reversal in adult females. Double mutants of _foxl2a_ and _foxl2b_

show complete sex reversal in the early stages of sexual maturation (Yang et al., 2017). Research on tilapia has shown that _foxl2_ directly regulates the transcription of _cyp19a1a_ (Wang

et al., 2007). During sex reversal in rice field eels, the expression levels of _cyp19a1a_ and _foxl2_ gradually decrease (Zhang et al., 2008; Zhang et al., 2013; Hu et al., 2014a), and DNA

methylation levels in the promoter regions of _cyp19a1a_ and _foxl2_ substantially increase (Wang et al., 2020). Studies have suggested that _cyp19a1a_ and _foxl2_ play important roles in

sex differentiation and sex reversal in rice field eels. In our previous study, the transcription activator-like effector nuclease (TALENs) technique was used to edit _cyp19a1a_ and _foxl2_

in rice field eels, and the efficiencies of a single target mutation were 87.5% and 58.3%, respectively (Feng et al., 2017). As _cyp19a1a_ and _foxl2_ were not completely knocked out using

this method, their functions are not well understood. This study aimed to develop an efficient multi-target gene editing method based on a platform for micromanipulation and genome editing

previously established in our laboratory. Rice field eels with approximately 100% mutation of _cyp19a1a_ and _foxl2_ were obtained by editing multiple target sites. The results showed that

the gonads of rice field eels without _cyp19a1a_ could not develop into ovaries, thereby indicating that _cyp19a1a_ plays a key role in regulating ovarian development in rice field eels.

SECTION SNIPPETS ANIMALS AND EMBRYOS The parent stocks of rice field eels were purchased from the Baishazhou Agricultural Market, Wuhan, China. Artificial spawning was performed according to

a previously established protocol (Feng et al., 2017). Briefly, female fish weighing 100–150 g were injected with 3 mg of carp pituitary, 10 μg of LHRH-A2, and 500 IU of HCG (Ningbo

Sansheng Pharmaceutical Co., Ltd., Ningbo, China) diluted in 1 ml 0.6% NaCl solution. Ovulation occurred approximately 40 h after injection. A small incision was MULTI-LOCUS EDITING OF

_CYP19A1A_ AND _FOXL2_ The _cyp19a1a_ gene of the rice field eel consists of nine exons. Seven targets in the first four exons of _cyp19a1a_ were chosen, and the corresponding gRNAs were

designed based on either the sense strand (a-e) or antisense strand (f-g) (Fig. 1A, Table S1). All seven gRNAs were mixed and co-injected with the Cas9 protein into the animal pole of

fertilized eggs (Fig. 1C). Once the injected egg developed into a late gastrulation stage embryo (60hpf), genomic DNA of the entire embryo was extracted DISCUSSION In this study, a highly

efficient gene editing method was established for the rice field eel, and individuals with 100% mutations in the target gene were obtained from the P0 generation. The _cyp19a1a_ and _foxl2_

genes of the rice field eel were successfully knocked out using multi-locus gene editing. In _cyp19a1a_ knockout fish, serum E2 levels were significantly decreased, and ovarian development

was blocked. In _foxl2_ knockout fish, _cyp19a1a_ expression decreased in the gonads, whereas _dmrt1_ AUTHOR CONTRIBUTIONS Wei Hu, Yanlong Song, and Mingxi Hou conceived and designed

research; Mingxi Hou,Yanlong Song, Ke Feng, Hongrui Luo, Yinjun Jiang, and Wen Xu performed the experiments; Ji Chen and Binbin Tao contributed new reagents and analytic tools; Yongming Li

provided resources; Wei Hu, Wei Hu, Yanlong Song, Mingxi Hou analyzed data and wrote the manuscript; Zuoyan Zhu revised the manuscript. FUNDING This study was supported by National Key

R&D Program of China (2018YFD0900203), China Agriculture Research System of MOF and MARA (CARS-46, NK2022010207), National Natural Science Foundation of China (32072961, 32102790), the

Foundation of Hubei (2022BBA0010) and Jiangshu Province (HSXT30311). DECLARATION OF COMPETING INTEREST The authors declare that they have no known competing financial interests or personal

relationships that might have influenced the work reported in this study. ACKNOWLEDGEMENTS We are grateful to Ms. Fang Zhou and Guangxin Wang (Analytical & Testing Center, IHB, CAS) for

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(10) * COMPARATIVE TRANSCRIPTOME ANALYSIS IDENTIFIED GENES INVOLVED IN OVARIAN DEVELOPMENT IN TAKIFUGU RUBRIPES 2025, Comparative Biochemistry and Physiology - Part D: Genomics and

Proteomics Show abstract Ovarian development is a complex process involving multiple genes, but the molecular mechanisms underlying this process in _Takifugu rubripes_ remain poorly

understood. This study aimed to identify genes associated with ovarian development in _T. rubripes_ and to investigate the regulatory mechanisms of oocyte maturation. Transcriptome data were

compared across four different developmental stages (stage II to V) to identify differentially expressed genes (DEGs) and perform GO and KEGG enrichment analysis. The expression patterns of

randomly selected genes were then validated by qPCR. The results yielded a total of 1,289,401,820 raw data from all libraries, with 16,929 DEGs identified across all comparison groups. The

DEGs were predominantly enriched in ovarian steroidogenesis, estrogen-mediated signaling, and TGF-beta signaling pathways. The qPCR analysis showed that _cyp17a1_ was identified as being

expressed at similar levels in stage II and III. Thereafter, _cyp17a1_ was observed to undergo a continuous increase in expression from stage III to V. _cyp19a1_, _nanos1_, _foxl2_ and _ar_

were identified as being expressed at similar levels at stage II and III, then increase in expression from stage III to IV and subsequent downregulation from stage IV to V. _hsd17b1_ was

identified as being expressed at similar levels at stage II and IV. This study represents a transcriptomic study of ovarian development in female _T. rubripes_. Several essential

ovarian-related genes and sex-related biological pathways were identified. The results will improve our understanding of the molecular mechanisms underlying ovarian development in this

species. * GENES FOR EDITING TO IMPROVE ECONOMIC TRAITS IN AQUACULTURE FISH SPECIES 2025, Aquaculture and Fisheries Citation Excerpt : In fighting fish (Betta splendens), the use of the

CRISPR/Cas9 system to knock out dmrt1 revealed that the absence of dmrt1 function leads to female development (Wang, Sun, et al., 2022). In the rice field eel (Monopterus albus), a

hermaphroditic fish, multi-locus gene editing successfully disrupted both cyp19a1a and foxl2 (Hou, et al., 2023). In mutants with a disrupted cyp19a1a gene, there was a notable decrease in

serum estrogen levels, leading to a halt in ovarian development. Show abstract Aquaculture, a critical sector for global food security, faces the challenge of meeting growing demand while

protecting wild fish populations. Gene editing, a powerful genetic tool, emerges as a potential solution. By modifying key genes in fish, it is expected to rapidly enhance growth rate,

disease resistance, and other economically important traits, leading to increased profitability, sustainability, and competitiveness of the industry. This review navigates the complex

landscape of genes for important traits, gene editing in aquaculture, exploring successes, challenges, and prospects for improved productivity, sustainability, and resilience. We provide

guidance for researchers and stakeholders in identifying and editing genes responsible for important traits, while addressing economic, environmental, regulatory, and ethical considerations.

This roadmap paves the way for a future where gene editing empowers aquaculture to meet global food needs while upholding environmental responsibility. * MULTI-OMICS ANALYSIS REVEALED THE

DYSFUNCTION OF OVARY AND TESTIS INDUCED BY CHRONIC HYPOXIA IN PELTEOBAGRUS FULVIDRACO 2024, Aquaculture Citation Excerpt : In contrast to the increased Foxl2 in the testes, hypoxic exposure

significantly downregulated Foxl2 transcription in the ovaries. The Foxl2 mutation results in the dysregulation of Cyp19a1a transcription and serum E2 levels in the ricefield eel (Monopterus

albus) and failure of ovary differentiation in XX female Nile Tilapia (Oreochromis niloticus) (Hou et al., 2023; Zhang et al., 2017a). Dysregulated transcripts of Foxl2 in both testes and

ovaries after chronic hypoxia exposure. Show abstract Hypoxia is a global problem that not only impairs parental fish reproductive functions and fertility but can also decrease offspring

fertility through the transgenerational effect, further resulting in the degradation of fish germplasm resources in aquaculture. However, an in-depth exploration of the comprehensive

mechanisms of hypoxia-induced gonadal impairment in teleosts is lacking. In this study, the juvenile yellow catfish (_Pelteobagrus fulvidraco_) were continuously exposed to hypoxic

conditions (1.9 ± 0.3 mg/L dissolved oxygen) for one month. The transcriptome, proteome, and metabolome were integrated to study the gonadal response to hypoxia in yellow catfish

comprehensively. Hypoxia altered gonadal biological processes involved in angiogenesis, energy metabolism, apoptosis, and steroidogenesis. The testes and ovaries exhibited different adaptive

mechanisms in response to chronic hypoxia. Angiogenesis was activated in the testes to increase oxygen delivery. Angiogenesis was inhibited in the ovaries when ovarian hemoglobin levels

increased. Activated glycolysis and catabolism of glycogenic amino acids were observed in the testes. The energy metabolism in the ovaries was disrupted by hypoxia, as reflected by

disordered glycolysis and suppressed synthesis of amino acids and fatty acids. Gonadal dysfunction and suppression of germ cell development by hypoxia were observed. The decrease in

synaptonemal complex protein 1 (SYCP1), SYCP3, Piwi like RNA-mediated gene silencing 2 (PIWIL2), and Nanos C2HC-type zinc finger 3 (_Nanos3)_ in the testes reflected suppressed meiosis and

spermatogenesis. The downregulation of vitellogenin 1 (VTG1), zona pellucida (ZP), growth differentiation factor 9 (GDF9), inhibin subunit beta B _(Inhbb)_, and insulin-like growth factor

(IGF) signaling inhibited folliculogenesis and vitellogenesis in the ovaries. Our results provide insight into the molecular mechanisms underlying hypoxia-induced reproductive impairment in

teleosts. * OPTIMISING COMMERCIAL TRAITS THROUGH GENE EDITING IN AQUACULTURE: STRATEGIES FOR ACCELERATING GENETIC IMPROVEMENT 2024, Reviews in Aquaculture * CYP19A1A PROMOTES OVARIAN

MATURATION THROUGH REGULATING E2 SYNTHESIS WITH ESTROGEN RECEPTOR 2A IN PAMPUS ARGENTEUS (EUPHRASEN, 1788) 2024, International Journal of Molecular Sciences * INDUCTION OF MEIOGYNOGENESIS IN

MONOPTERUS ALBUS AND GENETIC HOMOZYGOSITY ANALYSIS OF THE GYNOGENIC OFFSPRING 2024, Acta Hydrobiologica Sinica View all citing articles on Scopus View full text © 2022 Elsevier B.V. All

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