The global obesity epidemic has emerged as a major driver of cancer incidence and mortality, with accumulating evidence highlighting the gut-adipose-tumor axis as a critical mediator of obesity-related carcinogenesis. The gut-adipose-tumor axis is a tripartite communication network, wherein the intestinal microbiome, adipose tissue, and tumor microenvironment engage in dynamic bidirectional crosstalk that alters cancer susceptibility and progression. This review synthesizes current understanding of the epidemiology, pathophysiology, therapeutic implications, and future directions of this axis. Obesity-induced gut dysbiosis leads to systemic dissemination of pro-inflammatory microbial products and metabolites. These gut-derived signals profoundly influence adipose tissue homeostasis, exacerbating chronic low-grade inflammation, promoting macrophage infiltration and polarization, and disrupting adipokine secretion patterns. Dysfunctional adipose tissue generates cancer-promoting mediators and metabolic perturbations. The convergence of gut-derived and adipose-derived signals creates a systemic pro-carcinogenic environment that reshapes the tumor microenvironment through multiple mechanisms. Understanding the gut-adipose-tumor axis as an integrated biological system offers opportunities for cancer prevention and treatment. This is of significant importance for exploring the mechanisms of obesity-related carcinogenesis and developing new therapeutic approaches for obesity-related cancers.

By the integration of MGI short-read sequencing, PacBio HiFi long-read sequencing, and Hi-C techniques, we constructed a chromosome-scale genome assembly for Holothuria fuscocinerea, a highly abundant and widely distributed sea cucumber species in the family Holothuriidae. The assembly was anchored into 23 pseudochromosomes with a total size of 1.556 Gb after redundancy removal, demonstrating contig and scaffold N50 values of 5.18 Mb and 63.82 Mb, respectively, indicative of high contiguity. Genome quality assessment using Merqury and BUSCO analyses yielded a QV score of 60.4087 and a BUSCO completeness rate of 97.8%, confirming high assembly quality with minimal gaps. Among the 29,878 protein coding genes, 95 percent obtained functional annotations from at least one database. This high-resolution genome resource is expected to advance research on H. fuscocinerea and the Holothuroidea class, providing insights for population management, evolutionary studies, and genetics research.

Heliocidaris crassispina is an economically important sea urchin in the Northwest Pacific, yet large-scale aquaculture is constrained by high mortality during early development. This limitation is largely attributable to the poor understanding of gene expression patterns across these critical stages. Therefore, a stage-resolved developmental transcriptomic profile from fertilization to the settled juvenile is needed to clarify the molecular basis of early developmental transitions and to support improved hatchery management and stage-appropriate feeding. We described a complete developmental profile of H. crassispina from fertilization to the settled juvenile and established transcriptomes for 14 consecutive developmental stages. Beginning with the two-arm pluteus stage, H. crassispina undergoes rapid growth, paralleling a marked rise in mortality that continues through metamorphosis until the juvenile settles. Based on global gene expression patterns, development was divided into four periods. Period-I spanned the fertilized egg to 32-cell stages, Period-II covered two late-cleavage stages (LC1–LC2), Period-III extended from blastula to two-arm pluteus, and Period-IV extended from the four-arm pluteus to the settled juvenile stages. GO and KEGG analyses indicated a stepwise transition in gene expression programs across developmental periods, consistent with the morphological and physiological changes observed. Weighted gene co-expression network analysis (WGCNA) identified ten modules whose expression profiles correlated significantly with specific developmental periods, indicating development is regulated by a coordinated, module-level transcriptional program. Analysis of digestive enzyme and energy metabolism genes showed that early embryos rely on endogenous reserves, whereas feeding larvae progressively establish exogenous digestive capacity, accompanied by stage-dependent fluctuations and a late-stage rise in glycolysis and TCA cycle gene expression, suggesting a metabolic transition associated with exogenous feeding and settlement. This study established transcriptomes for 14 consecutive stages of H. crassispina from fertilization to the settled juvenile and, together with growth and survival, resolved four transcriptomic periods. Functional enrichment and WGCNA indicated coordinated, period-specific programs that track major morphological and physiological transitions. Digestive enzyme and energy metabolism genes showed a clear shift from reliance on endogenous reserves in embryos to the progressive establishment of exogenous digestive capacity in feeding larvae, accompanied by late-stage upregulation of glycolysis and TCA cycle genes around settlement. These insights support stage-specific feeding and hatchery management to reduce mortality.

Despite lacking a definitive visual organ, sea cucumbers clearly demonstrate an ability to perceive light. This study identifies the anterior oral tentacles, dorsal papillae, and ventral tube feet as the light-sensitive areas of the tropical sea cucumber Holothuria leucospilota. In H. leucospilota adults, only one active-site-containing opsin, specifically a rhabdomeric opsin (r-opsin), is expressed in the light-sensitive areas, thus providing an opportunity to characterize its photoreceptive structures through localization of this single gene. This r-opsin is functionally confirmed to be light-sensitive and capable of activating the Gq pathway. It exhibits a maximum absorption wavelength at 459 nm, which overlaps with the spectrum of blue light that triggers negative phototaxis responses in H. leucospilota, and it also mediates the responses to adjacent wavelengths. The r-opsin-expressing cells are distributed in the oral tentacles, papillae, and tube feet, forming distinct light-sensing structures with specialized morphologies. These structures are interconnected by nerve cords, establishing a comprehensive photoreception system throughout the body, which integrates the diffuse photoreception of pentaradial animals and the anterior photoreception seen in most bilateral animals. This study elucidates a unique sea cucumber photoreception system, shedding light on the last previously unexplored class within Echinodermata and offers evolutionary insights into deuterostome vision.

Gallic acid (GA) is a plant-derived polyphenol with antioxidant and antimicrobial activities, yet its efficacy as a functional feed additive in sea cucumbers remains unclear. Here, we systematically evaluated graded dietary GA supplementation in the tropical sea cucumber Holothuria leucospilota by integrating growth performance, body-wall nutritional composition, intestinal histomorphology, digestive and antioxidant enzyme activities, gut microbial community profiles, and intestinal transcriptomic responses. Sea cucumbers were randomly assigned to six dietary treatments containing 0, 200, 400, 800, 1600, or 3200 mg kg-1 GA and fed for 75 days. GA elicited a pronounced nonlinear, dose-dependent response. The intermediate dose (GA2; 400 mg kg-1) produced a relatively favorable response under the present feeding conditions, increasing final body weight and increasing the accumulation of crude protein and structural-protein-associated amino acids in the body wall, while preserving brush-border integrity and significantly enhancing α-amylase, lipase, cellulase, and superoxide dismutase (SOD) activities. Transcriptomic profiling revealed clear divergence between GA-treated groups and the control (Con), with GA2 characterized by upregulation of pathways associated with digestive hydrolysis, nutrient transport, lipid utilization, and redox/detoxification processes. Microbiome analyses showed progressive community restructuring with increasing GA, including reduced Proteobacteria and enrichment of Firmicutes/Bacilli at high doses, decreased α-diversity, and pronounced genus-level turnover. Correlation-based integration further resolved two host-microbe interaction modules that linked microbial taxa either to nutrient assimilation programs or to intestinal barrier and immune-response programs. Collectively, these findings support GA as a promising functional additive for H. leucospilota within a narrow optimal-dose window, while highlighting potential risks of dysbiosis and intestinal injury at excessive inclusion levels.

To date, the understanding of lectin-related immunity in sea cucumbers remains limited. Through a comprehensive genome-wide analysis, 133 lectin genes were identified from Holothuria leucospilota, a sea cucumber species with high ecological and economic significance in the Pacific and Indian Oceans. These lectins were classified into five subtypes, with C-type lectin being the predominant form, and the presence of SUEL-like lectin and Malectin reported here for the first time in sea cucumbers. Transcriptomic analysis across twelve tissues and sixteen developmental stages revealed ubiquitous expression of H. leucospilota lectins. A key finding was the compartmentalization of lectin systems: SUEL-like lectins expressed in the body wall constitute an “external” defense system, while C-type lectins produced by coelomocytes form an “internal” immune system. This spatial and functional specialization reflects a sophisticated immune strategy in invertebrates, enabling targeted pathogen recognition at different bodily interfaces. Furthermore, distinct immune responses to Vibrio, LPS, and poly(I:C) were observed among different lectins. We also identified an ovary-specific C-type lectin, suggesting a potential role in early embryonic development. This study not only fills a knowledge gap regarding lectin genes in sea cucumbers but also establishes a broader framework for understanding complex immune mechanisms in echinoderms.

With the rapid development of gene editing technology, its application in breast cancer has gradually become the focus of research. This article reviews the application of gene editing technology in the treatment of breast cancer, and discusses its challenges and future development directions. The key application areas of gene editing technology in the treatment of breast cancer will be outlined, including the discovery of new therapeutic targets and the development of drugs related to the pathway. Gene editing technology has played an important role in the discovery of new therapeutic targets. Through the use of gene editing technology, breast cancer-related genes are systematically edited to regulate key regulatory factors on related pathways or key tumor suppressor genes such as FOXC1 and BRCA, and the results are analyzed in cell or animal experiments, and the target is obtained from the experimental results, which provides important clues for the development of new drugs. This approach provides an innovative way to find more effective treatment strategies and inhibit tumor growth. In addition, gene editing technology has also promoted the personalization of breast cancer treatment. By analyzing a patient's genomic information, researchers can pinpoint key genetic mutations in a patient's tumor and design personalized treatments. This personalized treatment approach is expected to improve the therapeutic effect and reduce adverse reactions. Finally, the application of gene editing technology also provides support for the development of breast cancer immunotherapy. By editing immune cells to make them more potent against tumors, researchers are trying to develop more effective immunotherapies to bring new treatment options to breast cancer patients.

Methyl farnesoate (MF) serves as an essential regulator of key developmental processes in crustaceans, similar to juvenile hormones (JHs) in insects. However, it is susceptible to degradation in circulation. Despite the detection of proteins binding to MF in crustacean hemolymph for 3 decades, the precise genes encoding these proteins remain unclear. The present study identifies genes in crustaceans containing the juvenile hormone-binding protein (JHBP) domain. Among the 11 JHBPs found in the Pacific white shrimp (Litopenaeus vannamei), XP_027209752.1, which is primarily expressed in the hepatopancreas, emerges as the predominant form. This protein exhibits selective binding to MF rather than to JHs, leading to its designation as a methyl farnesoate-binding protein (MFBP) in crustaceans. Alterations in the amino acid sequence are predicted to induce structural changes that enhance the affinity of MFBP for MF. Endogenous MFBP inhibits molting, consistent with the function of MF. Furthermore, positive regulation of MFBP by MF has been observed in hepatopancreatic primary cells, with similar trends of hepatopancreatic MFBP mRNA and hemolymph MF levels during molting and ovarian development. This study identifies a novel MFBP in crustaceans that, despite being a homolog of insect JHBP, specifically binds MF to regulate molting in penaeid shrimp. These findings may advance our understanding of crustacean endocrine regulation and provide a molecular basis for improving their aquaculture techniques.

A chromosome-level genome assembly of Bohadschia ocellata, a member of the Holothuriidae family, was constructed through the integration of MGI DNBSEQ-T7 short-read sequencing, PacBio HiFi long-read sequencing, and Hi-C genomic scaffolding technology. After optimization to eliminate redundant sequences, the genome assembly was precisely anchored to 23 chromosomes, resulting in a total size of 909.18 Mb. The N50 of its contig and scaffold sequences were 12.00 Mb and 38.97 Mb, respectively, confirming that the assembly was highly continuous. According to Merqury and BUSCO evaluations, the genome assembly reached a QV of 64.44 and completeness of 94.40%. From this assembly, 31,277 protein-coding genes were identified, which were 98.10% complete based on BUSCO assessment of the predicted proteome. Functional annotations were obtained from at least one database for more than 99% of these genes. This high-quality B. ocellata genome assembly from the current study could offer valuable information for further genetic and evolutionary studies of this sea cucumber species.

Sea cucumbers are renowned for their regenerative capabilities, making them ideal models for studying tissue and organ regeneration. Holothuria leucospilota possesses a unique defensive structure, the Cuvierian organ (CO), which is ejected upon threat and regenerates within weeks. However, the molecular mechanisms underlying CO regeneration remain poorly understood. In this study, we induced CO expulsion in H. leucospilota by mechanical stimulation and examined the regeneration process over 31 days. Histological analysis revealed that regeneration initiated with mesothelium formation, followed by connective tissue and epithelium development. Transcriptomic analysis identified numerous differentially expressed genes during regeneration. Key extracellular matrix (ECM)-related genes were upregulated, while matrix protease genes were downregulated. Signaling pathways including Wnt and Hippo were suppressed, whereas apoptosis and cell cycle pathways were activated. Additionally, several structural outer-layer proteins showed altered expression. These results indicate that ECM reorganization and coordinated regulation of cell proliferation and apoptosis are central to CO regeneration. This study provides important insights into the molecular mechanisms of organ regeneration in echinoderms and offers valuable clues for regenerative studies in higher animals.

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms.

In this study, a chromosome-level genome of the tropical sea cucumber Stichopus monotuberculatus was generated by a combination of Nanopore long-read, Illumina short-read, and Hi-C sequencing technologies. The final assembly was 810.54 Mb in length, with contig N50 and scaffold N50 values of 10.15 Mb and 35.36 Mb, respectively. This assembly comprised 23 pseudo-chromosomes, covering 99.82% of the genome. Completeness analysis using BUSCO indicated that 97.8% of the metazoan conserved genes were presented in their entirety. A total of 29,596 protein-coding genes were predicted, with functional annotations available for 94.43% of these genes. The high-quality genome assembly produced in this study may provide an essential foundation for future researches on resource conservation and genetic breeding of S. monotuberculatus.

Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-β structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.

本文讨论了分子生物学课程在组学时代下面临的挑战以及如何进行有效教学.文章从课程内容、教学方法和策略以及教学改革等角度进行分析,并提出了一些具体建议,包括多层次的课程设置、注重信息获取与数据分析能力的培养、合理利用项目式教学、创设互动环节等.此外,培养学生的素质和社会责任感也是非常重要的.在推进分子生物学课程教育改革时,需要制定完善的教学方案、组建优秀的教师团队和实验室设备并协同多方力量.综上所述,本文为当前面对新挑战的分子生物学课程的教学提供了新思路和参考.

To date, studies on apoptosis in echinoderms mainly focus on the caspase-dependent extrinsic apoptotic pathway, while knowledges regarding the intrinsic apoptotic pathway is still limited. In this study, 55 genes belong to 7 families of the intrinsic apoptotic pathway, namely, Bcl-2, caspase, AIF, Cyt c, Apaf-1, Smac, HTRA2 and Endo G, were identified by screening of the genomic database of the tropical sea cucumber Holothuria leucospilota. The Bcl-2 family showed significant gene expansion in H. leucospilota but not in other echinoderms, while the pro-apoptotic factor Endo G is generally lost in echinoderms. Transcriptomic data showed that the genes of the intrinsic apoptotic pathway were ubiquitously expressed in all tissues, and all embryonic and larval developmental stages of H. leucospilota. The gene numbers and functional domains of the caspase family and AIF family were further clarified and showed conserved with their counterparts in vertebrates. In primary coelomocytes, the transcript expression of different caspases showed different responses to pathogenic challenges of vibrio and pathogen-associated molecular patterns, and environmental challenges of heave metal and reactive oxygen species. Overall, this study indicates that the intrinsic apoptotic pathway is conserved between vertebrates and echinoderms by using the sea cucumber H. leucospilota as a model. This study may contribute in increasing the knowledge of development and innate immunity in ancestral deuterostomia species.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5′ end splice donor and 3′ end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5′-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5′-GC) in the conserved sequence (5′-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.

Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-β, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

In mammals, leptin is an endocrine hormone involved in the regulation of feeding and energy metabolism, and glucocorticoids were previously reported to induce leptin gene expression. However, the link between glucocorticoids and leptin in fish has not been investigated. Goldfish are tetraploid teleosts because of genomic replication events (FSGD or 3-R), and some specific genes have quadrupled. In this study, two goldfish leptin-B genes, namely, leptin-BI (GenBank: ON468243) and leptin-BII (GenBank: ON468244), were cloned. The cDNA sequences of leptin-AI (GenBank: FJ534535.1) and leptin-AII (GenBank: FJ854572.1) have been reported by our team previously. Goldfish leptin-AI, -AII, -BI and -BII genes are located on chromosomes 43, 18, 29 and 4, respectively. In goldfish, leptin-B proteins shared approximately 30 % amino acid identities with leptin-A proteins and only 20 % with human leptin; however, either goldfish leptin-A or leptin-B showed high amino acid homology between themselves, 79.29 % and 82.74 %, respectively. The four leptin genes in goldfish are all highly conserved in three-dimensional (3-D) protein structures and gene structures. A tissue expression study showed that both the A and B forms of goldfish leptins are predominantly in the brain and liver. The effects of glucocorticoids on four leptin transcripts expression in goldfish were investigated by in vivo intraperitoneal injection and in vitro cell incubation approaches. The results showed that cortisol inhibited hepatic leptin-A transcript expression in a dose-dependent manner, while cortisol stimulated hepatic leptin-B mRNA expression, and RU486 (glucocorticoid receptor inhibitor) abolished cortisol’s effect on hepatic leptin-A and leptin-B mRNA expression. These results reflected the differential regulation of leptin-A and leptin-B by glucocorticoids in goldfish, which implied that leptin-A and leptin-B may mediate different functions in goldfish.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR ; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates the functions of interleukin-2 (IL-2) at the levels of transcription, splicing and translation, and plays other critical roles in the immune system. ILF2 is well-documented in vertebrates, while little is currently known in crustacean species such as the Pacific white shrimp (Litopenaeus vannamei). In the present study, five cDNA for spliced isoforms of Lv-ILF2 were identified, in which four of them are the full-length long isoforms (Lv-ILF2-L1, Lv-ILF2-L2, Lv-ILF2-L3 and Lv-ILF2-L4) and one of them is a truncated short isoform (Lv-ILF2-S). The whole sequence of ILF2 gene from L. vannamei was obtained, which is 11,680 bp in length with 9 exons separated by 8 introns. All five isoforms contain a domain associated with zinc fingers (DZF). Two alternative splicing types (alternative 5' splice site and alternative 3' splice site) were identified in the five isoforms. The Lv-ILF2 mRNA showed a broad distribution in all detected tissues, and the Lv-ILF2-L transcript levels were higher than those of Lv-ILF2-S in corresponding tissues. The mRNA levels of Lv-ILF2-S in the hepatopancreas, heart, muscle and stomach, but not in the eyestalk, were significantly increased after challenges with Vibrio harveyi or lipopolysaccharide (LPS), while no significant changes were observed for the transcript levels of Lv-ILF2-L in these tissues under the same immune stimulants. On the contrary, the transcript levels of neither Lv-ILF2-S nor Lv-ILF2-L were affected by challenges of polyinosinic: polycytidylic acid [Poly (I:C)]. In addition, after knockdown of the Lv-ILF2 mRNA level by siRNA, the mortality of shrimp and the hepatopancreatic bacterial numbers were significantly increased under V. harveyi challenge, indicating that Lv-ILF2 might participate in the immune defenses against V. harveyi invasion. Collectively, our study here supplied the first evidence for a novel splicing mechanism of ILF2 transcripts, and provided a functional link between the Lv-ILF2 isoforms and the capacity against pathogenic Vibrio in penaeid shrimp.