Highly Efficient Production of MSTN-Edited Hu Sheep Mediated by the CBE System

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Wang, W. J. Liu, C. H. Meng, H. L. Wang, Z. K. Cui, J. Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6928613/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Aug, 2025 Read the published version in Functional & Integrative Genomics → Version 1 posted 9 You are reading this latest preprint version Abstract The cytosine base editor (CBE) enables precise C-to-T substitution without DNA double-strand breaks, which offering a promising tool for editing livestock genomes to enhance economically valuable traits. In this study, using Hu sheep, characterized by high reproductive performance but suboptimal meat production as the research subject, two CBE-editing sgRNAs (sgM1 and sgM2) targeting the negative regulator MSTN (Myostatin) gene were designed. The results revealed a 75% editing efficiency of sgM2 at the parthenogenetically activated embryonic level and no detectable off-target effects. Thirty-four Hu sheep zygotes microinjected with sgM2 and CBE mRNA mixtures were transferred into four recipient ewes, yielding four lambs with confirmed MSTN editing and no off-target activity. Growth performance data revealed that MSTN -edited Hu sheep exhibited higher body weights at 120-180 days, and significantly enlarged muscle fiber cross-sectional areas compared to wild-type controls. Edited Hu sheep displayed reduced MSTN protein expression, elevated p-AKT levels, and diminished p-ERK and p-p38 signaling. In conclusion, MSTN -edited Hu sheep were highly efficient generated using CBE, and further analysis demonstrate that MSTN editing activates the AKT pathway while suppressing MAPK signaling, leading to muscle fiber hypertrophy and accelerated growth, which provides technical methodologies and breeding materials for developing fast-growing, meat-type Hu sheep strains. Hu sheep MSTN CBE off-target effects Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Implications The cytosine base editor (CBE) offers a promising tool for editing livestock genomes to enhance economically valuable traits. This study, focused on Hu sheep with high fecundity but suboptimal meat performance, and targeted the myostatin ( MSTN ) gene, successfully identified sgRNAs demonstrating 75% embryonic-stage editing efficiency in ovine embryos and further obtained 4 g0 gene-editing Hu sheep (100% efficiency) with no off-target activity utilizing cytosine base editor (CBE) technology. Gene-edited sheep demonstrated significantly higher body weight and muscle cross-sectional area compared to control group Hu sheep counterparts. Furthermore, the observed growth phenotypes were mediated through the MAPK and AKT signaling pathway. Introduction The Hu sheep, a native breed from China's Taihu Lake region, is characterized by high prolificacy, heat-humidity tolerance, and adaptability to confined feeding, making it a critical genetic resource for meat sheep production in China (Guo et al., 2023 ). However, its suboptimal meat yield remains a limitation. Myostatin ( MSTN ), also known as growth differentiation factor 8 ( GDF-8 ), belongs to the TGF-β superfamily (McPherron et al., 1997 ) and functions as a negative regulator of skeletal muscle growth (Gao et al., 2013 ; Hsu et al., 2014 ). Numerous studies demonstrate that MSTN mutations disrupt muscle development and carcass traits, inducing a "double-muscling" phenotype in cattle, sheep, and other species (Boman et al., 2009 ; Boman and Vage, 2009 ; Clop et al., 2006 ; Gill et al., 2009 ; Grobet et al., 1997 ; Grobet et al., 1998 ; Karim et al., 2000 ; McPherron et al., 1997 ; Mosher et al., 2007 ; Schuelke et al., 2004 ; Shelton and Engvall, 2007 ). Consequently, MSTN is expected to become a prime target for improving growth and carcass traits in Hu sheep. CRISPR-Cas systems provide a robust technical support for precise livestock genome editing to enhance economically valuable traits (Tait-Burkard et al., 2018 ). These systems enable rapid, efficient, and targeted gene mutagenesis (Cong et al., 2013 ), allowing stable inheritance of improved traits within two generations while preserving original breed advantages. CRISPR-Cas9 coupled with somatic cell nuclear transfer (SCNT) have been widely used to generate large animal models, including MSTN -edited pigs (Li et al., 2020 ; Peng et al., 2021 ; Zhu et al., 2020 ), MSTN -edited horses (Moro et al., 2020 ), CD46 -edited cattle (Workman et al., 2023 ), CFTR -edited sheep (Fan et al., 2018 ), and IFNAR -edited sheep (Davies et al., 2022 ). However, SCNT requires in vitro preparation of edited somatic cells, reconstruction of embryos, and subsequent embryo transfer - a complex, low-efficiency process with high resource demands (Matoba and Zhang, 2018 ). To reduce costs and improve efficiency, direct microinjection of Cas9 mRNA and sgRNA mixtures into single-cell zygotes has been employed (Guo et al., 2016 ; Guo et al., 2023 ; Kalds et al., 2022 ; Niu et al., 2014 ), yet this approach risks mosaicism, genotypic complexity, and challenges in progeny selection. To address these limitations, base editor (BE) systems - comprising deaminases, Cas9 variants, and sgRNAs - were developed based on CRISPR-Cas9 (Komor et al., 2016 ), which were classified as cytosine base editor (CBE) or adenine base editor (ABE) based on deaminase type. With its characteristics such as high precision, no generation of double-strand breaks (DSB), and relatively simple editing form, it has been widely applied in animal breeding. For instance, Zhu et al.(2022) used ABE to disrupt exon 6 of the porcine GHR gene, achieving the loss of the GHR gene at both the mRNA and protein levels; another study(Song et al., 2022 ) simultaneous editing of CD163 , MSTN and IGF2 via CBE enhanced growth and disease resistance in pigs; Zhou et al.(2019) induced the p.R96C mutation in ovine SOCS2 using BE3, yielding 25% editing efficiency and accelerated growth in mutant sheep. Additional studies targeted GDF9 (Xu et al., 2022 ) to increase lambing rates in sheep, while introducing premature stop codons in caprine FGF5 improved wool yield (Li et al., 2019 ). Notably, no studies have yet reported CBE-mediated MSTN editing in Hu sheep zygotes to generate MSTN -edited strains. Here, we propose using the CBE system to introduce premature stop codons in MSTN for targeted inactivation. Our study screened highly efficient and precise sgRNAs at the embryonic level, employed the CBE system to generate MSTN -edited Hu sheep, and evaluated phenotypic outcomes, MSTN protein expression, and downstream signaling pathways. This work aims to provide technical strategies and breeding materials for developing fast-growing, meat-type Hu sheep through gene editing. Materials and Methods Animals Hu sheep were maintained at the Luhe Animal Scientific Base of the Jiangsu Academy of Agricultural Sciences in Jiangsu province. The experimental procedures were approved by the Research Committee of the Jiangsu Academy of Agricultural Sciences and conducted with adherence to the Regulations for the Administration of Affairs Concerning Experimental Animals (Decree No. 63 of the Jiangsu Academy of Agricultural Science on 8 July 2014). Preparation of sgRNA and CBE mRNA Two sgRNAs targeting the ovine MSTN gene (Gene ID: 100125998) were designed using the CRISPOR online tool ( http://crispor.tefor.net/ ) (Supplementary Table 1). Annealed sgRNA oligonucleotides were cloned into the pGL3-U6-sgRNA-PGK-puromycin plasmid (addgene:51133) through BsaI restriction enzyme cites (Supplementary Fig. 1). A T7 promoter-containing primer (Supplementary Table 2) was used for PCR amplification with Phanta HS Super-Fidelity DNA Polymerase (P525, Vazyme, China) under the following conditions: 98°C for 3 min; 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for extension (1 min/kb); and a final extension at 72°C for 10 min. The PCR product was gel-purified (28104, Qiagen, China) to generate the sgRNA transcription template. In vitro transcription of sgRNA was performed using 200 ng of purified template with the MEGAshortscript™ T7 Transcription Kit (Am1354, Ambion, China). For BE3 mRNA synthesis, a T7 promoter-containing primer (Supplementary Table 2) was used to amplify the CMV-YE1-BE3-FNLS-CMV-mCherry plasmid (addgene:154005) template under identical PCR conditions. The resulting 1,000 ng of purified template was transcribed using the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Am1345, Ambion, China). RNA was purified and prepared by the RNA clean Beads (N412-02, Vazyme, China). RNA quality was assessed via agarose gel electrophoresis, and concentrations were quantified using a NanoDrop 2000 spectrophotometer. Embryo Microinjection and Editing Validation Parthenogenetically activated embryos were microinjected with 5 pL of a mixture containing 50 ng/µL sgRNA and 100 ng/µL BE3 mRNA. Post-injection, embryos were cultured to the blastocyst stage for editing validation. Whole-genome amplification of single blastocysts was performed using the Single-Cell Whole Genome Amplification Kit (N603-0, Vazyme, China). Target regions were PCR-amplified (primers in Supplementary Table 3) and sequenced via Sanger sequencing. Embryo Transfer Donor ewes underwent synchronized estrus and superovulation as described by Guo et al. ( 2023 ). The mixture of sgM2 and BE3 mRNA was injected into the cytoplasm of the zygote, with the injection volume and concentration being the same as those used in the in vitro embryo injection experiment, then cultured in G1 medium for 1 h, the well-developed zygotes after injection were selected and transplanted into the ampulla of the fallopian tubes of recipient ewes that were synchronized in estrus. Eight to nine embryos were transplanted into each recipient ewe. Genotyping and Off-Target Analysis Genomic DNA was extracted from lamb ear tissues using the phenol-chloroform method. Target regions were PCR-amplified and sequenced to confirm editing. Potential off-target sites (POTs) were predicted using Cas-OFFinder, and the top five sites with minimal mismatches were PCR-amplified (primers in Supplementary Table 4) and sequenced. g1 MSTN gene-editing Hu sheep preparation g0 homozygous MSTN gene-edited ram (R007) was mated with two wild-type ewes through natural mating, and produced 4 g1 individuals (two ewes and two rams). MSTN gene-editing Hu sheep Phenotypic Analysis g0 and g1 MSTN gene-editing Hu sheep and counterpart controls were raised under the same conditions. And the body weight was recorded every 60 days. At 6 months of age, the R007 and counterpart controls were anesthetized, then the posterior gluteal muscles were surgically collected for HE staining to evaluate muscle development. Western Blotting Posterior gluteal muscles tissues were homogenized in 200 µL RIPA lysis buffer (P0013B, Beyotime Biotechnology, Nantong, China) containing protease inhibitors. Protein concentrations were normalized using a BCA assay (P0010, Beyotime Biotechnology, Nantong, China). Samples (30 µg) were denatured at 95°C for 10 min, separated on 12.5% SDS-PAGE gels (80 V for 30 min, 120 V for 1 h), and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies against GAPDH (60004-1-Ig, Proteintech, Wuhan, China), p38 (9272S, Cell Signaling Technology), p-p38 (8690S, Cell Signaling Technology), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (4370T, Cell Signaling Technology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), and MSTN (19142-1-AP, Proteintech). Then, the corresponding secondary antibodies were added, and the expression levels of the proteins were detected using the ECL kit (Dallas, TX, USA) on the Image Quant LAS 4000 (Fuji Film, Tokyo, Japan). Band intensities were quantified using ImageJ (NIH, Bethesda, MA, USA). Statistical Analysis Data were expressed as mean ± standard error of mean (SEM). All analyses were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL, USA) and Graphpad Prism7 software (GraphPad Software, La Jolla, CA, USA). Differences between the two groups were determined using independent sample t-tests. Multiple comparisons were conducted using a one-way analysis of variance and Bonferroni test. Statistical significance was considered at P < 0.05 (*). Results High editing efficiency sgRNA screening at embryo level mediated by CBE Two specific sgRNAs, named sgM1 and sgM2, were designed targeting exon 1 and exon 3 of the MSTN gene, respectively (Fig. 1 -A). High-quality in vitro transcribed sgRNAs and BE3 mRNA showed clear bands of expected sizes (Fig. 1 B). Parthenogenetically activated embryos microinjected with sgM1 + BE3 or sgM2 + BE3 mRNA (n = 16 per group) were analyzed by PCR amplification and sanger sequencing. The results showed the C to T editing efficiency at the sgM2 target site was 75.0%, while no editing was found at the sgM1 target site (Fig. 1 -C and Table 1 ). Therefore, the sgM2 was selected for subsequent MSTN editing in Hu sheep. Table 1 Summary of sgRNA editing efficiency at the embryo level. sgRNA Total number Edit number Editing efficiency sgM1 16 0 0/16(0%) sgM2 16 12 12/16(75%) Production of MSTN-edited Hu sheep mediated by CBE A total of 58 fertilized eggs were collected from five donor ewes, 34 viable embryos were microinjected with sgM2 and CBE mRNA and transplanted into four recipient ewes, and one recipient ewe successfully gave birth to four lambs: #R007, #R009, #R012 and #R014 (Table 2 ). Among them, lamb #R014 died immediately after birth, and lamb #R009 succumbed to trampling at six days postpartum, the other two lambs (#R007 and R012) survived. Table 2 Summary of the production of MSTN -edited sheep using CBE. sgRNA Donor Superovulated Embryo Injected/ Embryos Transferred Delivered Recipients /Total Recipients Edited Lambs /Total Lambs sgM2 5 58/34 1/4(25%) 4/4(100%) Genotyping analysis revealed that lamb #R007 exhibited homozygous editing at the target site, while lambs #R009, #R012, and #R014 showed mosaic editing (Fig. 2 -B). Additionally, #R014 carried two distinct forms of long-fragment deletions (Fig. 2 -C). Off-target analysis at the individual level Off-target analysis revealed no mutations at the five predicted potential off-target sites in individuals edited at the MSTN sgM2 site, which suggesting that no off-target events occurred in these gene edited Hu sheep (Fig. 3 ). Phenotypic Analysis of MSTN-gene Edited Hu Sheep The body weight of 2 g0 (#R007 and #R012) and 4 g1 generation gene-edited sheep from D0 to D180 were recorded (Fig. 4 -A and 4 -B), the genotyping results of g1 generation gene-edited Hu sheep are shown in Supplementary Fig. 2. The results showed that the edited Hu sheep exhibited significantly heavier body weights than WT from D120 to D180 (p < 0.05) (Fig. 4 -A). Compared with WT, the edited sheep showed a more pronounced phenotype in the front leg and backward muscles tissue, with a distinct “double muscling” phenotype (Fig. 4 -B). HE-stained sections of the posterior gluteal muscles (Fig. 4 -C) showed significantly larger muscle fiber cross-sectional areas in MSTN -KO sheep (P < 0.01). MSTN and downstream signaling pathways analysis in MSTN-edited Hu Sheep To determine the effects of MSTN -edited on MSTN protein, MSTN and several downstream signaling regulators (AKT, ERK1/2, and P38) were selected for protein detection (Fig. 5 ). In MSTN homozygous edited Hu sheep, MSTN protein was nearly absent, while mosaic edited sheep showed reduced expression. In MSTN homozygous edited sheep, p-AKT was upregulated, while total AKT expression were increased in mosaic edited sheep. The expression of ERK was downregulated in all MSTN edited sheep, with p-ERK downregulated in homozygous edited sheep. Additionally, the expression of p-P38 was downregulated in both homozygous and mosaic edited sheep compared to wild-type individuals. Discussion The Hu sheep is a vital genetic resource for meat production in China; however, its suboptimal meat yield remains a limiting factor. To address this, our study employed the CRISPR-Cas9-derived CBE system to efficiently generate MSTN -edited Hu sheep. All four g0 lambs exhibited editing (100% efficiency), marking the first report of such high C-to-T conversion efficiency in sheep genome editing. Notably, adult MSTN -edited lambs displayed a pronounced "double-muscling" phenotype, demonstrating the feasibility and potential of CBE systems for livestock genome editing. The CBE system (Komor et al., 2016 ), which comprises a cytidine deaminase fused to catalytically inactive Cas9 (dCas9), enables sgRNA-guided C-to-T conversions within a narrow editing window without double-strand breaks. Owing to this precision, CBE holds significant promise for livestock breeding. However, reported editing efficiencies vary substantially across studies. For example, Zhou et al. ( 2019 ) utilized a base editor to introduce the p.R96C mutation in ovine SOCS2 with 25.0% efficiency, whereas Xu et al. ( 2022 ) employed BE4-Gam to edit the GDF9 locus (p.S395F), improving prolificacy traits in sheep at 16.3% efficiency. Notably, our study successfully generated MSTN -edited Hu sheep via CBE with 100% editing efficiency. Crucially, CBE editing efficiency is primarily determined by sgRNA design (Huszar et al., 2023 ) and editor selection. On the one hand, significant efficiency variations exist among different sgRNAs targeting the same gene. As demonstrated by Li et al ( 2019 ), BE3-mediated introduction of premature stop codons in caprine FGF5 exon 1: only sgRNA1 exhibited an effective on target editing efficiency of 9% among four candidate sgRNAs, while the other three showed minimal activity, highlighting sgRNA-dependent variability in BE3 efficiency. Another study used the BE4-Gam to edit the GDF9 gene without sgRNA screening yielded only 16.3% efficiency (Xu et al., 2022 ). In this study, to achieve high editing efficiency of sgRNA, we designed two sgRNAs targeting MSTN and identified sgM2 with 75% editing efficiency in ovine parthenogenetic embryos, which serve as an efficient and economical model for predicting in vivo gene editing outcomes (Freking and Leymaster, 2006 ), allowing indirect evaluation of editing efficiency at the individual level. Furthermore, four lambs generated via microinjection combined with embryo transfer were all edited with an efficiency of 100%; on the other hand, the high editing efficiency is also related to the use of YE1-BE3-FNLS, a highly efficient editor, which has a preference for editing sgRNA PAM distal 6–7 nt (Zuo et al. 2020 ), and our sgM2 precisely conforms to this preference feature. Compared to the CRISPR-Cas9 system, CBE’s narrow editing window enables precise single-base substitutions with reduced genotypic complexity (Abeuova et al., 2023 ; Dyke et al., 2023 ; Jeong et al., 2022 ) and lower off-target events (Kim et al., 2017 ). He et al. ( 2018 ) employed Cas9 mRNA coupled with a single-guide RNA targeting MSTN (sgRNA-1) to generate five g0 goats through NHEJ-dependent editing. All animals showed mosaic genotypes with six unique deletion patterns and zero homozygous edits. In this study, homozygotes accounted for 25% of the four g0 lambs, the predominant editing pattern was C-to-T conversion. Moreover, four g1 lambs produced by mating R007 with wild-type Hu sheep are all heterozygous, and the genotype is stably inherited, which is beneficial for reducing the breeding cycle for obtaining homozygous offspring. Regarding off-target effects, some researches indicated that sgRNA binding to homologous sequences induces off-target activity (Tian et al., 2023 ; Wienert and Cromer, 2022 ; Xu et al., 2023 ), which are less frequent in BE systems than in CRISPR/Cas9 (Kim et al., 2017 ). Consistent with prior studies (Zhou et al., 2019 ; Li et al., 2019 ), no off-target activity was detected in the four MSTN -edited lambs in this study. Therefore, CBE tool can be applied to precision breeding of sheep. The MSTN -edited Hu sheep exhibited hypertrophic musculature, aligning with phenotypes observed in natural MSTN mutants (Boman et al., 2011 ; Clop et al., 2006 ; Mosher et al., 2007 ; Wang et al., 2018 ) and knockout models (Guo et al., 2016 ; Guo et al., 2023 ; Wang et al., 2016 ). Histological analysis revealed increased muscle fiber diameter and cross-sectional area in the posterior gluteal muscles, which consistent with MSTN loss triggering muscle hyperplasia (Fan et al., 2022 ; Guo et al., 2016 ; McPherron et al., 1997 ). MSTN -edited Hu sheep demonstrated significantly (P < 0.05) greater body weights than wild-type controls at both 4- and 6-month developmental stages. These findings are consistent with the known effects of MSTN knockout on the growth performance of Hu sheep and align with the conclusions of other studies that MSTN knockout enhances body weight (Crispo et al., 2015 ; Guo et al., 2023 ; Proudfoot et al., 2015 ; Zhou et al., 2022 ). MSTN , a member of the TGF-β superfamily, functions as a negative regulator of skeletal muscle development by inhibiting cellular proliferation and differentiation. The molecular mechanism involves MSTN binding to the activin type IIB receptor (ActRIIB) on muscle cells, which triggers Smad2/3-dependent signaling to suppress AKT phosphorylation (Bataille et al., 2021 ). Notably, this inhibitory effect is physiologically reversible - studies in cultured myoblasts demonstrate that elevated AKT activation can overcome MSTN-mediated suppression, thereby promoting myogenic differentiation and myotube hypertrophy (Trendelenburg et al., 2009 ). In vivo, MSTN overexpression reduces muscle mass via AKT suppression (Sartori et al., 2009 ), whereas MSTN deficiency elevates AKT expression (Morissette et al., 2009 ). Our results align with this paradigm, showing upregulated p-AKT in homozygous MSTN -KO Hu sheep, while increased total AKT levels in mosaic knockouts showed. Additionally, the MAPK/ERK pathway is also critical for cell differentiation and anti-apoptosis (Sun et al., 2015 ), which is regulated by ERK1/2, a key mediator of cell proliferation (Joneson and Bar-Sagi, 1997 ). In C2C12 cells, MSTN promotes ERK1/2 activation during proliferation and differentiation (Yang et al., 2006 ). In our study, MSTN knockout downregulated ERK1/2, potentially contributing to muscle fiber hypertrophy in edited sheep. Moreover, inhibition of p38 activity alleviates the proliferation suppression of mouse embryonic fibroblasts caused by MSTN overexpression (Philip et al., 2005 ), and our results indicated that MSTN -knockout Hu sheep exhibited reduced expression of p-p38, which may accelerate cells proliferation and thereby promote muscle growth. In summary, this study establishes CBE as a highly efficient platform for generating MSTN -edited Hu sheep. Demonstrating that the edited sheep exhibited activated AKT signaling, suppressed MAPK pathways, and consequent muscle hypertrophy, and accelerated growth, underscoring CBE’s potential for advancing precision livestock breeding. Declarations Funding This study was supported by Jiangsu Seed Industry Vitalization and Open Competition Project (JBGS〔2021〕025). Competing interests The authors declare no competing interests. Author Contribution Y. Wang: Writing – original draft, Methodology, Visualization. W. J. Liu: Writing – original draft, Methodology, Visualization, software. C. H. Meng: Data curation, Software, Methodology; H.L. Wang: Supervision, Writing-review & editing, Investigation; Z.K. Cui: Methodology; J. Zhang: Methodology, Visualization, Project administration; J.L. Zhang: Methodology; Y. Qian: Resources, Supervision; Y.X. Li: Data curation, Validation, Visualization, Resources, Writing – review &editing; S.X. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6928613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":477769447,"identity":"9dfd805d-a3fe-43dd-9f9a-90d837ee2473","order_by":0,"name":"Y. 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Zhang","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"L.","lastName":"Zhang","suffix":""},{"id":477769454,"identity":"b5ce16bc-66b2-4dad-ac83-a4330e0e7ca0","order_by":7,"name":"Y. Qian","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Y.","middleName":"","lastName":"Qian","suffix":""},{"id":477769455,"identity":"3f9b37f0-0cfb-400c-b01b-620f1f8925b2","order_by":8,"name":"Y. X. Li","email":"","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Y.","middleName":"X.","lastName":"Li","suffix":""},{"id":477769459,"identity":"140dfa32-6a2b-46c1-b5a0-282e6550d8f4","order_by":9,"name":"S. X. Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYNCCCjaStZwhWQtjGymq5WckP/zwcR6fPH//AcYPPxjs8ghqMThzzFhy5jY2wxk3EpglexiSiwlrYe9hkObdxsbYcIOBQZqB4UBiA0GHNfMw/+adw2Y///wB5t9EaWE43sMmzdvAlrjhQAIbcbYA/WJmOeMYW/LGG4ltlj0GyUQ4bEby4xsfao7Zzjt/+PCNHxV2RDgMAo4BMSNQsQGR6oGghnilo2AUjIJRMPIAAAx3OaPLSviiAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"S.","middleName":"X.","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2025-06-19 07:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6928613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6928613/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10142-025-01698-8","type":"published","date":"2025-08-27T15:56:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85651850,"identity":"751ede5c-4857-45fd-bc9b-b9dd78c0b1d7","added_by":"auto","created_at":"2025-06-30 09:30:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93451,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of sgRNA editing efficiency at embryo level. \u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of sgRNA target sites in the \u003cem\u003eMSTN\u003c/em\u003e gene of Hu sheep. \u003cstrong\u003eB\u003c/strong\u003e In vitro transcription of sgRNA and BE3 mRNA. \u003cstrong\u003eC\u003c/strong\u003e Editing efficiency analysis of sgRNA at the embryo level.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/82bff9caa1eb04b3b7a944ca.png"},{"id":85652211,"identity":"ae26165f-a1be-4983-be9a-3985a410702b","added_by":"auto","created_at":"2025-06-30 09:38:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":183099,"visible":true,"origin":"","legend":"\u003cp\u003eGenome editing analysis in lambs. \u003cstrong\u003eA\u003c/strong\u003eThe PCR amplification of the sgM2 target site. \u003cstrong\u003eB\u003c/strong\u003e Genotyping sequencing detection. \u003cstrong\u003eC\u003c/strong\u003e Long fragment deletion analysis in lamb #R014.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/b1b265a2ea8336bdf2e53fe4.png"},{"id":85651855,"identity":"ffb66b5e-015d-4d0f-a6d2-133535edebe6","added_by":"auto","created_at":"2025-06-30 09:30:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":197627,"visible":true,"origin":"","legend":"\u003cp\u003eSequencing analysis of \u003cem\u003eMSTN\u003c/em\u003e-sgM2 POTs at the individual level. \u003cstrong\u003eA\u003c/strong\u003e wild type (WT); \u003cstrong\u003eB\u003c/strong\u003e Edited sheep.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/ac48a2fa17b046f715167c93.png"},{"id":85651854,"identity":"5e920d24-a968-46c6-a997-6d1a50ee9187","added_by":"auto","created_at":"2025-06-30 09:30:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":245594,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic analysis of \u003cem\u003eMSTN\u003c/em\u003e-edited Hu Sheep. \u003cstrong\u003eA\u003c/strong\u003e Body weight statistics of from D0 to D180. \u003cstrong\u003eB\u003c/strong\u003e Double muscling phenotype in edited Hu sheep. \u003cstrong\u003eC\u003c/strong\u003e HE-stained of posterior gluteal muscles.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/0e8b8a171427cb411c4747ee.png"},{"id":85652212,"identity":"3899d15a-74b2-4e73-a70c-0a3ff545f435","added_by":"auto","created_at":"2025-06-30 09:38:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":73440,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of MSTN and other proteins in \u003cem\u003eMSTN\u003c/em\u003e-KO Hu sheep compared to wild-type. \u003cstrong\u003eA\u003c/strong\u003e Protein expression at the individual level in wild-type and \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep. \u003cstrong\u003eB\u003c/strong\u003e Statistical analysis of protein expression at the individual level in wild-type and \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/324cbdc5bdb1db43741a11a7.png"},{"id":90345618,"identity":"67fb7bb3-9573-4b25-bd8b-65f4111de65b","added_by":"auto","created_at":"2025-09-01 16:10:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/6f16f1a7-f162-4a3f-8594-851d67422590.pdf"},{"id":85651852,"identity":"6c2f6eab-cecf-46f9-bcc1-555a3772b486","added_by":"auto","created_at":"2025-06-30 09:30:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3026208,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/15d01cca9fed4259ffa746e9.docx"},{"id":85651860,"identity":"ac76b381-5397-4bf9-82bb-5040526f1a63","added_by":"auto","created_at":"2025-06-30 09:30:22","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3298253,"visible":true,"origin":"","legend":"","description":"","filename":"thefulluncroppedGelsandBlotsimages.rar","url":"https://assets-eu.researchsquare.com/files/rs-6928613/v1/64485bc1bfbf14c1120db6fe.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Highly Efficient Production of MSTN-Edited Hu Sheep Mediated by the CBE System","fulltext":[{"header":"Implications","content":"\u003cp\u003eThe cytosine base editor (CBE) offers a promising tool for editing livestock genomes to enhance economically valuable traits. This study, focused on Hu sheep with high fecundity but suboptimal meat performance, and targeted the myostatin (\u003cem\u003eMSTN\u003c/em\u003e) gene, successfully identified sgRNAs demonstrating 75% embryonic-stage editing efficiency in ovine embryos and further obtained 4 g0 gene-editing Hu sheep (100% efficiency) with no off-target activity utilizing cytosine base editor (CBE) technology. Gene-edited sheep demonstrated significantly higher body weight and muscle cross-sectional area compared to control group Hu sheep counterparts. Furthermore, the observed growth phenotypes were mediated through the MAPK and AKT signaling pathway.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe Hu sheep, a native breed from China's Taihu Lake region, is characterized by high prolificacy, heat-humidity tolerance, and adaptability to confined feeding, making it a critical genetic resource for meat sheep production in China (Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, its suboptimal meat yield remains a limitation.\u003c/p\u003e \u003cp\u003eMyostatin (\u003cem\u003eMSTN\u003c/em\u003e), also known as growth differentiation factor 8 (\u003cem\u003eGDF-8\u003c/em\u003e), belongs to the TGF-β superfamily (McPherron et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and functions as a negative regulator of skeletal muscle growth (Gao et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hsu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Numerous studies demonstrate that \u003cem\u003eMSTN\u003c/em\u003e mutations disrupt muscle development and carcass traits, inducing a \"double-muscling\" phenotype in cattle, sheep, and other species (Boman et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Boman and Vage, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Clop et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Gill et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Grobet et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Grobet et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Karim et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; McPherron et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Mosher et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Schuelke et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Shelton and Engvall, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Consequently, \u003cem\u003eMSTN\u003c/em\u003e is expected to become a prime target for improving growth and carcass traits in Hu sheep.\u003c/p\u003e \u003cp\u003eCRISPR-Cas systems provide a robust technical support for precise livestock genome editing to enhance economically valuable traits (Tait-Burkard et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These systems enable rapid, efficient, and targeted gene mutagenesis (Cong et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), allowing stable inheritance of improved traits within two generations while preserving original breed advantages. CRISPR-Cas9 coupled with somatic cell nuclear transfer (SCNT) have been widely used to generate large animal models, including \u003cem\u003eMSTN\u003c/em\u003e-edited pigs (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eMSTN\u003c/em\u003e-edited horses (Moro et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eCD46\u003c/em\u003e-edited cattle (Workman et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), \u003cem\u003eCFTR\u003c/em\u003e-edited sheep (Fan et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and \u003cem\u003eIFNAR\u003c/em\u003e-edited sheep (Davies et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, SCNT requires in vitro preparation of edited somatic cells, reconstruction of embryos, and subsequent embryo transfer - a complex, low-efficiency process with high resource demands (Matoba and Zhang, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo reduce costs and improve efficiency, direct microinjection of Cas9 mRNA and sgRNA mixtures into single-cell zygotes has been employed (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kalds et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Niu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), yet this approach risks mosaicism, genotypic complexity, and challenges in progeny selection. To address these limitations, base editor (BE) systems - comprising deaminases, Cas9 variants, and sgRNAs - were developed based on CRISPR-Cas9 (Komor et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which were classified as cytosine base editor (CBE) or adenine base editor (ABE) based on deaminase type. With its characteristics such as high precision, no generation of double-strand breaks (DSB), and relatively simple editing form, it has been widely applied in animal breeding. For instance, Zhu et al.(2022) used ABE to disrupt exon 6 of the porcine \u003cem\u003eGHR\u003c/em\u003e gene, achieving the loss of the \u003cem\u003eGHR\u003c/em\u003e gene at both the mRNA and protein levels; another study(Song et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) simultaneous editing of \u003cem\u003eCD163\u003c/em\u003e, \u003cem\u003eMSTN\u003c/em\u003e and \u003cem\u003eIGF2\u003c/em\u003e via CBE enhanced growth and disease resistance in pigs; Zhou et al.(2019) induced the p.R96C mutation in ovine \u003cem\u003eSOCS2\u003c/em\u003e using BE3, yielding 25% editing efficiency and accelerated growth in mutant sheep. Additional studies targeted \u003cem\u003eGDF9\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) to increase lambing rates in sheep, while introducing premature stop codons in caprine \u003cem\u003eFGF5\u003c/em\u003e improved wool yield (Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, no studies have yet reported CBE-mediated \u003cem\u003eMSTN\u003c/em\u003e editing in Hu sheep zygotes to generate \u003cem\u003eMSTN\u003c/em\u003e-edited strains. Here, we propose using the CBE system to introduce premature stop codons in \u003cem\u003eMSTN\u003c/em\u003e for targeted inactivation. Our study screened highly efficient and precise sgRNAs at the embryonic level, employed the CBE system to generate \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep, and evaluated phenotypic outcomes, MSTN protein expression, and downstream signaling pathways. This work aims to provide technical strategies and breeding materials for developing fast-growing, meat-type Hu sheep through gene editing.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eHu sheep were maintained at the Luhe Animal Scientific Base of the Jiangsu Academy of Agricultural Sciences in Jiangsu province. The experimental procedures were approved by the Research Committee of the Jiangsu Academy of Agricultural Sciences and conducted with adherence to the Regulations for the Administration of Affairs Concerning Experimental Animals (Decree No. 63 of the Jiangsu Academy of Agricultural Science on 8 July 2014).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of sgRNA and CBE mRNA\u003c/h3\u003e\n\u003cp\u003eTwo sgRNAs targeting the ovine \u003cem\u003eMSTN\u003c/em\u003e gene (Gene ID: 100125998) were designed using the CRISPOR online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Supplementary Table\u0026nbsp;1). Annealed sgRNA oligonucleotides were cloned into the pGL3-U6-sgRNA-PGK-puromycin plasmid (addgene:51133) through BsaI restriction enzyme cites (Supplementary Fig.\u0026nbsp;1). A T7 promoter-containing primer (Supplementary Table\u0026nbsp;2) was used for PCR amplification with Phanta HS Super-Fidelity DNA Polymerase (P525, Vazyme, China) under the following conditions: 98\u0026deg;C for 3 min; 35 cycles of 98\u0026deg;C for 10 s, 60\u0026deg;C for 30 s, and 72\u0026deg;C for extension (1 min/kb); and a final extension at 72\u0026deg;C for 10 min. The PCR product was gel-purified (28104, Qiagen, China) to generate the sgRNA transcription template. In \u003cem\u003evitro\u003c/em\u003e transcription of sgRNA was performed using 200 ng of purified template with the MEGAshortscript\u0026trade; T7 Transcription Kit (Am1354, Ambion, China). For BE3 mRNA synthesis, a T7 promoter-containing primer (Supplementary Table\u0026nbsp;2) was used to amplify the CMV-YE1-BE3-FNLS-CMV-mCherry plasmid (addgene:154005) template under identical PCR conditions. The resulting 1,000 ng of purified template was transcribed using the mMESSAGE mMACHINE\u0026trade; T7 ULTRA Transcription Kit (Am1345, Ambion, China). RNA was purified and prepared by the RNA clean Beads (N412-02, Vazyme, China). RNA quality was assessed via agarose gel electrophoresis, and concentrations were quantified using a NanoDrop 2000 spectrophotometer.\u003c/p\u003e\n\u003ch3\u003eEmbryo Microinjection and Editing Validation\u003c/h3\u003e\n\u003cp\u003eParthenogenetically activated embryos were microinjected with 5 pL of a mixture containing 50 ng/\u0026micro;L sgRNA and 100 ng/\u0026micro;L BE3 mRNA. Post-injection, embryos were cultured to the blastocyst stage for editing validation. Whole-genome amplification of single blastocysts was performed using the Single-Cell Whole Genome Amplification Kit (N603-0, Vazyme, China). Target regions were PCR-amplified (primers in Supplementary Table\u0026nbsp;3) and sequenced via Sanger sequencing.\u003c/p\u003e\n\u003ch3\u003eEmbryo Transfer\u003c/h3\u003e\n\u003cp\u003eDonor ewes underwent synchronized estrus and superovulation as described by Guo et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The mixture of sgM2 and BE3 mRNA was injected into the cytoplasm of the zygote, with the injection volume and concentration being the same as those used in the in vitro embryo injection experiment, then cultured in G1 medium for 1 h, the well-developed zygotes after injection were selected and transplanted into the ampulla of the fallopian tubes of recipient ewes that were synchronized in estrus. Eight to nine embryos were transplanted into each recipient ewe.\u003c/p\u003e\n\u003ch3\u003eGenotyping and Off-Target Analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from lamb ear tissues using the phenol-chloroform method. Target regions were PCR-amplified and sequenced to confirm editing. Potential off-target sites (POTs) were predicted using Cas-OFFinder, and the top five sites with minimal mismatches were PCR-amplified (primers in Supplementary Table\u0026nbsp;4) and sequenced.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eg1 MSTN gene-editing Hu sheep preparation\u003c/h2\u003e \u003cp\u003eg0 homozygous \u003cem\u003eMSTN\u003c/em\u003e gene-edited ram (R007) was mated with two wild-type ewes through natural mating, and produced 4 g1 individuals (two ewes and two rams).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMSTN gene-editing Hu sheep Phenotypic Analysis\u003c/h3\u003e\n\u003cp\u003eg0 and g1 \u003cem\u003eMSTN\u003c/em\u003e gene-editing Hu sheep and counterpart controls were raised under the same conditions. And the body weight was recorded every 60 days. At 6 months of age, the R007 and counterpart controls were anesthetized, then the posterior gluteal muscles were surgically collected for HE staining to evaluate muscle development.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003ePosterior gluteal muscles tissues were homogenized in 200 \u0026micro;L RIPA lysis buffer (P0013B, Beyotime Biotechnology, Nantong, China) containing protease inhibitors. Protein concentrations were normalized using a BCA assay (P0010, Beyotime Biotechnology, Nantong, China). Samples (30 \u0026micro;g) were denatured at 95\u0026deg;C for 10 min, separated on 12.5% SDS-PAGE gels (80 V for 30 min, 120 V for 1 h), and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4\u0026deg;C with primary antibodies against GAPDH (60004-1-Ig, Proteintech, Wuhan, China), p38 (9272S, Cell Signaling Technology), p-p38 (8690S, Cell Signaling Technology), ERK1/2 (4695T, Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2 (4370T, Cell Signaling Technology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), and MSTN (19142-1-AP, Proteintech). Then, the corresponding secondary antibodies were added, and the expression levels of the proteins were detected using the ECL kit (Dallas, TX, USA) on the Image Quant LAS 4000 (Fuji Film, Tokyo, Japan). Band intensities were quantified using ImageJ (NIH, Bethesda, MA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). All analyses were performed using SPSS (version 16.0; SPSS Inc., Chicago, IL, USA) and Graphpad Prism7 software (GraphPad Software, La Jolla, CA, USA). Differences between the two groups were determined using independent sample t-tests. Multiple comparisons were conducted using a one-way analysis of variance and Bonferroni test. Statistical significance was considered at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHigh editing efficiency sgRNA screening at embryo level mediated by CBE\u003c/h2\u003e \u003cp\u003eTwo specific sgRNAs, named sgM1 and sgM2, were designed targeting exon 1 and exon 3 of the \u003cem\u003eMSTN\u003c/em\u003e gene, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-A). High-quality in vitro transcribed sgRNAs and BE3 mRNA showed clear bands of expected sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Parthenogenetically activated embryos microinjected with sgM1\u0026thinsp;+\u0026thinsp;BE3 or sgM2\u0026thinsp;+\u0026thinsp;BE3 mRNA (n\u0026thinsp;=\u0026thinsp;16 per group) were analyzed by PCR amplification and sanger sequencing. The results showed the C to T editing efficiency at the sgM2 target site was 75.0%, while no editing was found at the sgM1 target site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-C and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, the sgM2 was selected for subsequent \u003cem\u003eMSTN\u003c/em\u003e editing in Hu sheep.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of sgRNA editing efficiency at the embryo level.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEdit number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEditing efficiency\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/16(0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12/16(75%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProduction of MSTN-edited Hu sheep mediated by CBE\u003c/h2\u003e \u003cp\u003eA total of 58 fertilized eggs were collected from five donor ewes, 34 viable embryos were microinjected with sgM2 and CBE mRNA and transplanted into four recipient ewes, and one recipient ewe successfully gave birth to four lambs: #R007, #R009, #R012 and #R014 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among them, lamb #R014 died immediately after birth, and lamb #R009 succumbed to trampling at six days postpartum, the other two lambs (#R007 and R012) survived.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the production of \u003cem\u003eMSTN\u003c/em\u003e-edited sheep using CBE.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgRNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDonor\u003c/p\u003e \u003cp\u003eSuperovulated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmbryo Injected/\u003c/p\u003e \u003cp\u003eEmbryos Transferred\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDelivered Recipients\u003c/p\u003e \u003cp\u003e/Total Recipients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEdited Lambs\u003c/p\u003e \u003cp\u003e/Total Lambs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esgM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58/34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/4(25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/4(100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGenotyping analysis revealed that lamb #R007 exhibited homozygous editing at the target site, while lambs #R009, #R012, and #R014 showed mosaic editing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-B). Additionally, #R014 carried two distinct forms of long-fragment deletions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOff-target analysis at the individual level\u003c/h2\u003e \u003cp\u003eOff-target analysis revealed no mutations at the five predicted potential off-target sites in individuals edited at the \u003cem\u003eMSTN\u003c/em\u003e sgM2 site, which suggesting that no off-target events occurred in these gene edited Hu sheep (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic Analysis of MSTN-gene Edited Hu Sheep\u003c/h2\u003e \u003cp\u003eThe body weight of 2 g0 (#R007 and #R012) and 4 g1 generation gene-edited sheep from D0 to D180 were recorded (Fig.\u0026nbsp;\u0026lt;link rid=\"fig4\"\u0026gt;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u0026lt;/link\u0026gt;\u003c/span\u003e-A and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B), the genotyping results of g1 generation gene-edited Hu sheep are shown in Supplementary Fig.\u0026nbsp;2. The results showed that the edited Hu sheep exhibited significantly heavier body weights than WT from D120 to D180 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A). Compared with WT, the edited sheep showed a more pronounced phenotype in the front leg and backward muscles tissue, with a distinct \u0026ldquo;double muscling\u0026rdquo; phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-B). HE-stained sections of the posterior gluteal muscles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-C) showed significantly larger muscle fiber cross-sectional areas in \u003cem\u003eMSTN\u003c/em\u003e-KO sheep (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMSTN and downstream signaling pathways analysis in MSTN-edited Hu Sheep\u003c/h2\u003e \u003cp\u003eTo determine the effects of \u003cem\u003eMSTN\u003c/em\u003e-edited on MSTN protein, MSTN and several downstream signaling regulators (AKT, ERK1/2, and P38) were selected for protein detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In \u003cem\u003eMSTN\u003c/em\u003e homozygous edited Hu sheep, MSTN protein was nearly absent, while mosaic edited sheep showed reduced expression. In \u003cem\u003eMSTN\u003c/em\u003e homozygous edited sheep, p-AKT was upregulated, while total AKT expression were increased in mosaic edited sheep. The expression of ERK was downregulated in all \u003cem\u003eMSTN\u003c/em\u003e edited sheep, with p-ERK downregulated in homozygous edited sheep. Additionally, the expression of p-P38 was downregulated in both homozygous and mosaic edited sheep compared to wild-type individuals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Hu sheep is a vital genetic resource for meat production in China; however, its suboptimal meat yield remains a limiting factor. To address this, our study employed the CRISPR-Cas9-derived CBE system to efficiently generate \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep. All four g0 lambs exhibited editing (100% efficiency), marking the first report of such high C-to-T conversion efficiency in sheep genome editing. Notably, adult \u003cem\u003eMSTN\u003c/em\u003e-edited lambs displayed a pronounced \"double-muscling\" phenotype, demonstrating the feasibility and potential of CBE systems for livestock genome editing.\u003c/p\u003e \u003cp\u003eThe CBE system (Komor et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which comprises a cytidine deaminase fused to catalytically inactive Cas9 (dCas9), enables sgRNA-guided C-to-T conversions within a narrow editing window without double-strand breaks. Owing to this precision, CBE holds significant promise for livestock breeding. However, reported editing efficiencies vary substantially across studies. For example, Zhou et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) utilized a base editor to introduce the p.R96C mutation in ovine \u003cem\u003eSOCS2\u003c/em\u003e with 25.0% efficiency, whereas Xu et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) employed BE4-Gam to edit the \u003cem\u003eGDF9\u003c/em\u003e locus (p.S395F), improving prolificacy traits in sheep at 16.3% efficiency. Notably, our study successfully generated \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep via CBE with 100% editing efficiency.\u003c/p\u003e \u003cp\u003eCrucially, CBE editing efficiency is primarily determined by sgRNA design (Huszar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and editor selection. On the one hand, significant efficiency variations exist among different sgRNAs targeting the same gene. As demonstrated by Li et al (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), BE3-mediated introduction of premature stop codons in caprine \u003cem\u003eFGF5\u003c/em\u003e exon 1: only sgRNA1 exhibited an effective on target editing efficiency of 9% among four candidate sgRNAs, while the other three showed minimal activity, highlighting sgRNA-dependent variability in BE3 efficiency. Another study used the BE4-Gam to edit the \u003cem\u003eGDF9\u003c/em\u003e gene without sgRNA screening yielded only 16.3% efficiency (Xu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, to achieve high editing efficiency of sgRNA, we designed two sgRNAs targeting \u003cem\u003eMSTN\u003c/em\u003e and identified sgM2 with 75% editing efficiency in ovine parthenogenetic embryos, which serve as an efficient and economical model for predicting in vivo gene editing outcomes (Freking and Leymaster, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), allowing indirect evaluation of editing efficiency at the individual level. Furthermore, four lambs generated via microinjection combined with embryo transfer were all edited with an efficiency of 100%; on the other hand, the high editing efficiency is also related to the use of YE1-BE3-FNLS, a highly efficient editor, which has a preference for editing sgRNA PAM distal 6\u0026ndash;7 nt (Zuo et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and our sgM2 precisely conforms to this preference feature.\u003c/p\u003e \u003cp\u003eCompared to the CRISPR-Cas9 system, CBE\u0026rsquo;s narrow editing window enables precise single-base substitutions with reduced genotypic complexity (Abeuova et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dyke et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jeong et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and lower off-target events (Kim et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). He et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) employed Cas9 mRNA coupled with a single-guide RNA targeting \u003cem\u003eMSTN\u003c/em\u003e (sgRNA-1) to generate five g0 goats through NHEJ-dependent editing. All animals showed mosaic genotypes with six unique deletion patterns and zero homozygous edits. In this study, homozygotes accounted for 25% of the four g0 lambs, the predominant editing pattern was C-to-T conversion. Moreover, four g1 lambs produced by mating R007 with wild-type Hu sheep are all heterozygous, and the genotype is stably inherited, which is beneficial for reducing the breeding cycle for obtaining homozygous offspring. Regarding off-target effects, some researches indicated that sgRNA binding to homologous sequences induces off-target activity (Tian et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wienert and Cromer, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which are less frequent in BE systems than in CRISPR/Cas9 (Kim et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Consistent with prior studies (Zhou et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), no off-target activity was detected in the four \u003cem\u003eMSTN\u003c/em\u003e-edited lambs in this study. Therefore, CBE tool can be applied to precision breeding of sheep.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep exhibited hypertrophic musculature, aligning with phenotypes observed in natural \u003cem\u003eMSTN\u003c/em\u003e mutants (Boman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Clop et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mosher et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and knockout models (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Histological analysis revealed increased muscle fiber diameter and cross-sectional area in the posterior gluteal muscles, which consistent with \u003cem\u003eMSTN\u003c/em\u003e loss triggering muscle hyperplasia (Fan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; McPherron et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep demonstrated significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) greater body weights than wild-type controls at both 4- and 6-month developmental stages. These findings are consistent with the known effects of \u003cem\u003eMSTN\u003c/em\u003e knockout on the growth performance of Hu sheep and align with the conclusions of other studies that \u003cem\u003eMSTN\u003c/em\u003e knockout enhances body weight (Crispo et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Proudfoot et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMSTN\u003c/em\u003e, a member of the TGF-β superfamily, functions as a negative regulator of skeletal muscle development by inhibiting cellular proliferation and differentiation. The molecular mechanism involves MSTN binding to the activin type IIB receptor (ActRIIB) on muscle cells, which triggers Smad2/3-dependent signaling to suppress AKT phosphorylation (Bataille et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, this inhibitory effect is physiologically reversible - studies in cultured myoblasts demonstrate that elevated AKT activation can overcome MSTN-mediated suppression, thereby promoting myogenic differentiation and myotube hypertrophy (Trendelenburg et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In vivo, MSTN overexpression reduces muscle mass via AKT suppression (Sartori et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), whereas MSTN deficiency elevates AKT expression (Morissette et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our results align with this paradigm, showing upregulated p-AKT in homozygous \u003cem\u003eMSTN\u003c/em\u003e-KO Hu sheep, while increased total AKT levels in mosaic knockouts showed. Additionally, the MAPK/ERK pathway is also critical for cell differentiation and anti-apoptosis (Sun et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which is regulated by ERK1/2, a key mediator of cell proliferation (Joneson and Bar-Sagi, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In C2C12 cells, MSTN promotes ERK1/2 activation during proliferation and differentiation (Yang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In our study, \u003cem\u003eMSTN\u003c/em\u003e knockout downregulated ERK1/2, potentially contributing to muscle fiber hypertrophy in edited sheep. Moreover, inhibition of p38 activity alleviates the proliferation suppression of mouse embryonic fibroblasts caused by MSTN overexpression (Philip et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and our results indicated that \u003cem\u003eMSTN\u003c/em\u003e-knockout Hu sheep exhibited reduced expression of p-p38, which may accelerate cells proliferation and thereby promote muscle growth.\u003c/p\u003e \u003cp\u003eIn summary, this study establishes CBE as a highly efficient platform for generating \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep. Demonstrating that the edited sheep exhibited activated AKT signaling, suppressed MAPK pathways, and consequent muscle hypertrophy, and accelerated growth, underscoring CBE\u0026rsquo;s potential for advancing precision livestock breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by Jiangsu Seed Industry Vitalization and Open Competition Project (JBGS〔2021〕025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Wang: Writing \u0026ndash; original draft, Methodology, Visualization. W. J. Liu: Writing \u0026ndash; original draft, Methodology, Visualization, software. C. H. Meng: Data curation, Software, Methodology; H.L. Wang: Supervision, Writing-review \u0026amp; editing, Investigation; Z.K. Cui: Methodology; J. Zhang: Methodology, Visualization, Project administration; J.L. Zhang: Methodology; Y. Qian: Resources, Supervision; Y.X. Li: Data curation, Validation, Visualization, Resources, Writing \u0026ndash; review \u0026amp;editing; S.X. Cao: Investigation, Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision, Writing \u0026ndash; review \u0026amp;editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbeuova, L., Kali, B., Tussipkan, D., Akhmetollayeva, A., Ramankulov, Y., Manabayeva, S., 2023. CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato. 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A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat Methods 17(6):600-604. doi: 10.1038/s41592-020-0832-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hu sheep, MSTN, CBE, off-target effects","lastPublishedDoi":"10.21203/rs.3.rs-6928613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6928613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cytosine base editor (CBE) enables precise C-to-T substitution without DNA double-strand breaks, which offering a promising tool for editing livestock genomes to enhance economically valuable traits. In this study, using Hu sheep, characterized by high reproductive performance but suboptimal meat production as the research subject, two CBE-editing sgRNAs (sgM1 and sgM2) targeting the negative regulator \u003cem\u003eMSTN\u003c/em\u003e (Myostatin) gene were designed. The results revealed a 75% editing efficiency of sgM2 at the parthenogenetically activated embryonic level and no detectable off-target effects. Thirty-four Hu sheep zygotes microinjected with sgM2 and CBE mRNA mixtures were transferred into four recipient ewes, yielding four lambs with confirmed \u003cem\u003eMSTN\u003c/em\u003e editing and no off-target activity. Growth performance data revealed that \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep exhibited higher body weights at 120-180 days, and significantly enlarged muscle fiber cross-sectional areas compared to wild-type controls. Edited Hu sheep displayed reduced MSTN protein expression, elevated p-AKT levels, and diminished p-ERK and p-p38 signaling. In conclusion, \u003cem\u003eMSTN\u003c/em\u003e-edited Hu sheep were highly efficient generated using CBE, and further analysis demonstrate that \u003cem\u003eMSTN \u003c/em\u003eediting activates the \u003cem\u003eAKT\u003c/em\u003e pathway while suppressing \u003cem\u003eMAPK\u003c/em\u003e signaling, leading to muscle fiber hypertrophy and accelerated growth, which provides technical methodologies and breeding materials for developing fast-growing, meat-type Hu sheep strains.\u003c/p\u003e","manuscriptTitle":"Highly Efficient Production of MSTN-Edited Hu Sheep Mediated by the CBE System","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 09:30:17","doi":"10.21203/rs.3.rs-6928613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-17T00:48:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T07:25:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T06:21:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188973945762295583335508840434044370408","date":"2025-06-28T04:56:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200896975938373944321967243140479855311","date":"2025-06-26T01:06:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-25T23:11:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-23T22:39:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-23T22:38:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2025-06-19T07:35:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"970b9909-b2c0-4508-b9d0-595398906dc1","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:09:30+00:00","versionOfRecord":{"articleIdentity":"rs-6928613","link":"https://doi.org/10.1007/s10142-025-01698-8","journal":{"identity":"functional-and-integrative-genomics","isVorOnly":false,"title":"Functional \u0026 Integrative Genomics"},"publishedOn":"2025-08-27 15:56:59","publishedOnDateReadable":"August 27th, 2025"},"versionCreatedAt":"2025-06-30 09:30:17","video":"","vorDoi":"10.1007/s10142-025-01698-8","vorDoiUrl":"https://doi.org/10.1007/s10142-025-01698-8","workflowStages":[]},"version":"v1","identity":"rs-6928613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6928613","identity":"rs-6928613","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}