Genome-wide off-target assessment on the arginine vasotocin receptor V1a2 knockout Scomber japonicus (chub mackerel)

Genome-wide off-target assessment on the arginine vasotocin receptor V1a2 knockout Scomber japonicus (chub mackerel) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide off-target assessment on the arginine vasotocin receptor V1a2 knockout Scomber japonicus (chub mackerel) Ryo Takeuchi, Kaoru Ohno, Tetsushi Sakuma, Hirofumi Ohga, Naoki Nagano, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7219894/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background In recent decades, genome-edited fish production is gaining momentum among aquaculturists, but uncertainties about off-target mutations is a major concern for commercialization of genome-edited fish products. Recently, a knockout (KO) Scomber japonicus (chub mackerel) strain of arginine vasotocin receptor V1a2 ( V1a2 ) was developed using TALEN to reduce the cannibalism frequency of fry. Aside from this behavioral trait, the KO fish had no other phenotypic differences with their wild counterpart. Whether the changed trait is an outcome of unwanted mutation in the genome or not, is a prerequisite for consumer’s safety and psychological satisfaction and thus needs careful genome-wide analysis. Therefore, the objective of this study was to detect and evaluate potential off-target sites in the genome-edited V1a2 -KO chub mackerel strain. Results In the V1a2 -KO strain, approximately 0.9 million variants were identified through specific variant extraction. To assess potential off-target effects, putative mutagenesis sites for TALEN were predicted using the chub mackerel reference genome (available at NCBI), and the top 1,000 sites with lowest binding scores (indicating stronger predicted binding affinity) were selected. Comparison of these 1,000 predicted TALEN binding sites with ~ 0.9 million variants revealed 24 overlapping sites. Among them, only one site, corresponding to the target gene V1a2 (13-base deletion), having the lowest binding score of 12. 76, was located within a coding sequence (CDS), suggesting no phenotypic side effects in the V1a2 -KO strain. Additional, expected number of variants analysis confirmed that the remaining observed variations might be an outcome of spontaneous mutation. Conclusion Using genome-wide assessment and comparison with predicted TALEN binding sites, of genome-edited individuals with their close and distant relatives, we have identified 24 probable sites, among which only one, the 13 base deletion was located in the target gene’s CDS strongly indicating no off-target associated phenotypic changes. Cumulatively, our results confirms the precision of TALEN genome-editing system and safety of TALEN genome-edited products and strongly raises the possibility of commercialization of V1a2 -KO chub mackerel strain in future. Chub mackerel TALEN Off-target mutation Double strand break Sequence homology Figures Figure 1 Figure 2 Introduction Growing population and its associated food demand are expected to create a protein crisis and according to food and agriculture organization of the United Nations (FAO) fish culture especially aquaculture of improved varieties are potentially feasible way out [1]. On the other hand, capture fisheries (i.e., fish caught directly from the wild) have become stagnant, showing no signs of recovery [2], thereby further emphasizing the need for sustainable aquaculture of suitable fish strains and innovative fish breeding technologies. Generally, selective breeding is the most practiced method to elevate the performance and strain production, but recent advances in genome-editing technology made them a formidable choice to develop strains of desirable traits [3, 4, 5, 6]. While selective breeding requires a long time and considerable financial resources, genome-editing demands-controlled laboratory environments, comprehensive genomic data, and advanced technical expertise. Over the past decades, genome-editing tools such as ZFN, TALEN, and CRISPR/Cas systems which induce double strand breaks (DSBs) in genomic DNA to generate functional mutations (knockout, KO) [7, 8, 9, 10] have emerged as widely preferred and alternative technologies for sustainable food production. These genome-editing tools have contributed greatly to solving genetic diseases, analyzing gene function across crop plants and farmed animals [11, 12] and improve productivity. The success of genome-edited products has even encourages policy makers to rethink and even legalize genome-edited crops and fishes for mass propagation and consumptions [13]. For instance, myostatin -KO red sea bream [14] and a GABA-enriched tomato [15] are being approved for mass production and consumption in Japan. While genome-editing tools are attracting attention as a next-generation aquatic breeding system, one concern is the introduction of unwanted mutations (off-target mutations) by genome-editing tools [16, 17, 18, 19]. Genome-editing tools can be engineered to make extremely well-defined alterations to the intended target genomic locus, but one potential complication is that the engineered nuclease will create other, various degree of unintended genomic changes i.e. “off-target” activity due to imperfect specificity. Proof of the absence of genome-wide off-target mutations is necessary to dispel psychological concerns [20, 21]. Recently, our research team has also developed a docile chub mackerel with reduced cannibalism by mutating of arginine vasotocin receptor V1a2 (hereafter referred as V1a2 - KO) by TALEN in the Scomber japonicus (chub mackerel), a marine aquaculture model [22]. Apart from low aggression and reduced cannibalism, this knockout strain does not exhibit any other phenotypic differences compared to wild counterparts and thus expected to greatly contribute chub mackerel seed production. As a common marine fish inhabiting temperate and subtropical waters, genome-edited mackerel represents a promising candidate for enhancing the sustainability of marine aquaculture. Therefore, elucidating off-target effects is of paramount importance to ensure the confidence of both producers and consumers. In this study, to ascertain the safety issue, and evaluate the presence of unwanted mutation or genome-integration, we performed genome-wide off-target prediction and assessment to confirm whether serious mutations occur in V1a2 -KO chub mackerel produced by genome-editing. Materials & Methods Experimental Fish maintenance: All of the chub mackerel used in the experiment were raised in the P1A marine aquarium facility of ABRIC Karatsu Satellite of Kyushu University. The V1a2 -KO strain chub mackerel was knocked out as shown in our previous study [22]. F0 generation of the V1a2 -KO strain chub mackerel was produced in 2017, F1 heterozygous progenies were prepared by crossing the potential F0 adult mutant males with chub mackerel females purchased from aquaculture firm in Oita (Japan), while the F2 and F3 were produced by crossing heterozygous V1a2 mutants the genome was extracted from the F3 generation V1a2 -KO strain chub mackerel were used in this study. Our own chub mackerel strain (Q-saba: https://www.city.karatsu.lg.jp/page/1431.html) were reared in the same facility and F7 generation was used in this investigation. Fish breeding and rearing were performed using previously published protocols [22, 23]. DNA library preparation and analysis: High quality DNA were collected from gills. using Qiagen blood and tissue DNA isolation kit following manufacturer’s instructions. DNA libraries were prepared using standard Illumina DNA library preparation protocol and sequenced using Illumina short read sequencing (Table. 2). 9 sample groups covering a total of 4 V1a2- KO homozygous fish (2/ sex) and 49 cultured strains (Q-saba) (Table.1). Table 1. Sample name list. Sample Details V1a2- KO-F1 KO strain F3 generation Female, 1 sample V1a2 -KO-F5 KO strain F3 generation Female, 1 sample V1a2 -KO-M1 KO strain F3 generation Male, 1 sample V1a2 -KO-M4 KO strain F3 generation Male, 1 sample Qsaba-Female Farmed chub mackerel female, for genome-estimation Qsaba-F-pool Farmed chub mackerel female group, 12 samples pool Qsaba-M-pool Farmed chub mackerel male group, 12 samples pool Qsaba-L-pool Farmed chub mackerel large group, 12 samples pool Qsaba-S-pool Farmed chub mackerel small group, 12 samples pool Estimation of TALEN-mutagenesis sites: TALEN induces DSB by preparing two sets of complexes containing a nuclease (FokI) (Fig 2). The optimal theoretical conditions for TALEN-based DNA editing are that the TALEN dimer must bind to the genomic DNA and that the spacer sequence should be 12-24 bases in length [24]. The TALEN pair (left, 5ʹ-TTCCGCTTCTATGGTCCA-3ʹ and right, 5ʹ-TGGAGGTGTTTGACTAT-3ʹ), having a spacer of 14 bp, was used to target the S–S binding site of V1a2 and create the KO strain. In this study, potential TALEN mutagenesis sites were identified using Paired Target Finder (https://tale-nt.cac.cornell.edu/node/add/talef-off-paired) [25] in accordance with the previous reports [26, 27]. The GCF_027409825.1_fScoJap1.pri_genomic.fna.gz (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/) were as used as the reference genome database [28]. The RVD sequences were provided as follows; RVD sequence 1, NG HD HD NN HD NG NG HD NG NI NG NN NN NG HD HD NI NG; RVD sequence 2, NN NN NI NN NN NG NN NG NG NG NN NI HD NG NI NG. Minimum and maximum spacer lengths were set to 10 and 25, respectively. Other parameters were set as recommended. From the list of candidate mutagenesis sites, the top 1,000 sites with the lowest combined TAL1 and TAL2 scores were selected for downstream analysis, as these were expected to represent the highest binding specificity. Raw reads processing and variant calling: The raw read data were processed by fastp (v0.24.0) [29] and mapped to the chub mackerel genome (NCBI) as a reference genome by snap-aligner (v2.0.3) [30]. Low-quality reads were removed by ngsutils (v0.5.9, -mismatch 4 -properpair) [31]. Tentative variant calls and SNPs/INDELs filtering were then performed by GATK4 [32]. Variant calls by GATK4 (v4.6.1) were performed on all samples. The resulting SNPs/INDELs were treated as known sites and mutations were extracted by GATK4 once again (https://gatk.broadinstitute.org/hc/en-us/sections/360007226651-Best-Practices-Workflows). The bam file created was visualized by iGV (v2.18.2) [33], whenever necessary. Extraction of common and specific mutations in the V1a2 -KO strain chub mackerel: After variants calling, the resulting vcf file and bcftools (v1.21) [34] were used to extract variants common to the V1a2 -KO strain. Additional filtering was performed by removing the variants common to both V1a2 and Q-saba samples. Common variants were compared to DSB candidate sites (1,000 sites) by TALEN. The sites where the location of these variants overlapped with DSB candidate sites were treated as potential off-target-mutations sites. Further, the position of the mutation was compared to the position of the spacer sequence, and if the mutation existed within the spacer sequence, it was evaluated as a mutation that could have been caused by TALEN. Physiological assessment of off target effects: The potential off target sites were evaluated by aligning the individual sites to annotation database of chub mackerel (gtf format file: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/GCF_027409825.1_fScoJap1.pri_genomic.gtf.gz). The annotated genes, if any, were identified and evaluated further. Particularly, occurrence of frameshift mutation, changes in amino acid sequences and intronic and exonic mutation (changing the overall gene structure) were carefully checked. Results TALEN binding site listing Genome-editing through TALEN, CRISPR/Cas9 or other techniques creates DSB, but their activity and feasibility depends on the quantitative measurement of both on-target and off-target activity. Unlike the pseudo-random nature of retro or lenti-viral integration sites, nuclease off-target activity is presumed to be a direct result of the nuclease binding a nearby sequence with some level of homology to the intended target, and subsequently inducing a DSB. The elements that define the specificity of TALEN are the Left/Right TALEN motif sequence, DNA binding strength, and spacer length (Fig. 2). In this analysis, these were parameterized and adjusted to estimate the sites where DSB may have occurred. Among the top 1,000 candidate sites with the lowest binding scores, the site with the highest predicted binding affinity (i.e., the lowest total score), corresponded to the intended target: NC_070582.1, TAL1: 15,732,984; TAL2: 15,733,031, with a total score of 12.76. The second-ranked site was located at NC_070583.1, TAL1: 6,575,345; TAL2: 6,575,398, with a total score of 26.5, which is more than twice the score of the target site. Off-target evaluation by V1a2 -KO strain-specific variants and TALEN binding sites Whole-genome resequencing using Illumina short-read seq produced between 137,905,856 and 780,774,690 reads, of which 67,232,147 to 458,545,005 reads were successfully mapped to the chub mackerel reference genome (Table 2). Variant calling using GATK4 for the four V1a2 -KO strains identified approximately 7.0 to 7.7 million variants per sample, corresponding to a variation rate of 9.7–11.0%. In comparison, the control Q-saba samples exhibited 2.5–11.3% variation, with a total of 8,258,318 to 11,528,230 variants (Table 2). However, only 3,959,178 variants were found to be common across all four V1a2 -KO individuals. Importantly, a 13-base deletion in the V1a2 , the intended genome-editing target, was consistently detected in all four V1a2 -KO individuals (Fig. 1). Notably, both the V1a2 -KO and Q-saba strains used in this study originated from Oita, Japan, whereas the reference genome was established from a Korean population. Although chub mackerel is a highly migratory species, genetic polymorphisms arising from geographic isolation cannot be completely ruled out. To address this, we compared the NGS variant profiles of the V1a2 -KO and Q-saba strains maintained under identical laboratory conditions in Saga, Japan. Of the 3,959,178 shared variants observed in the V1a2 -KO strain, 896,196 variants (22.64%) were unique to this strain and not detected in Q-saba. The remaining variants were shared between the two groups (Table 1). To assess whether these V1a2 -KO-specific variants may be attributable to TALEN-mediated off-target effects, we compared them against the previously identified top 1,000 mutagenesis candidate sites. This comparison revealed 24 overlapping sites, including the intended V1a2 target gene site. For the remaining 23 overlapping sites (excluding the intended V1a2 target) comparison with publicly available gene annotations revealed no overlap with CDSs or intragenic introns (Table 3, Supplementary data). In other words, no evidence of TALEN-induced disruptions in protein-coding regions was found, and no unintended protein synthesis abnormalities were inferred outside of the V1a2 gene. Table 2 . Details of mutations in each sample (View Tables file) Table 3. Details of 23 off-target sites and V1a2 (View Tables file) Discussion Proper evaluation of genome-edited food products for off-target effects are of utmost importance to assess the consequences of genome-editing on both species physiology and consumer health. In this study, we conducted the first comprehensive evaluation of genome-editing effects in a marine teleost fish by using whole-genome sequencing data from the F3 generation of the V1a2 -KO chub mackerel. We first identified 1,000 potential mutagenesis candidate sites across the genome based on TALEN binding predictions, and then detected 24 strain-specific variants present only in the V1a2 -KO lineage. Finally, we assessed whether these 24 variants were caused by TALEN-mediated genome-editing or were spontaneous in origin. TALEN is widely used for genome-editing in plants to humans and infect [35], the first genome-edited crop, genome-edited soybean [36], that has reached the market was also generated using TALEN. TALEN requires a longer sequence length specification (30–40 bp) compared to other well-known genome-editing tools such as ZFN (9–18 bp) and CRISPR/Cas system (20 bp + PAM) [37, 38, 39] and thus, are predicted to be one of highly-specific genome-editing tools. We have used TALEN mRNA for the development of the V1a2 mutant and successfully obtained a specific mutant line with 13 base deletion [22]. Unlike previous reports [40], so far, we were unable to identify any genomic integration of reverse transcribed TALEN mRNAs highlighting the possibility of commercialization of this product. In plants, TALEN has been successfully employed to efficiently modify multiple gene copies simultaneously, for example, 107 out of 109 copies and alleles of the caffeic acid O-methyltransferase ( COMT ) gene in sugarcane were edited without any visible phenotypic defects [41, 42]. Notably, fish genomes have undergone whole-genome duplication during evolution and often contain two to four copies of the same gene. These characteristic makes TALEN a promising tool for generating food-grade, off-target-free genome-edited fish in a relatively short time. However, in present investigation, only a single TALEN pair targeting a single gene copy was used, and only one mutant line was analyzed. This limited scope restricts our ability to fully assess nuclease binding efficiency, the most reliable prediction methods for target site selection, and species-specific genome-editing dynamics and thus warrants further investigations evaluate the broader applicability of TALEN in fish species. Genetic mutations are often overrepresented in the inbreed strains [43, 44] and thus off-target assessment should consider comparing parents and progeny, wild and cultured strains, and geographically isolated population. In the studied V1a2 -KO strain, 3,062,982 out of 3,959,178 variants (77.4%) were found to be shared with the Q-saba strain. This high degree of overlap suggests substantial genetic diversity between the reference chub mackerel genome (derived from a Korean population) and the Oita-derived strains ( V1a2 -KO and Q-saba) used in this study. The observed differences may reflect underlying population effects, as the reference genome originated from South Korea [26], whereas both the V1a2 -KO and Q-saba strains were developed from fish collected in Oita, Japan. Geographic separation can lead to genetic diversification between populations, including the formation of distinct haplotypes, SNPs and INDELs [45, 46]. Whether such inter-population genetic differences can influence genome-editing efficiency or outcomes remains unclear due to limited available data and require further investigation in future. Additionally, genetic mutations may have been accumulated during domestication and selective breeding processes [47]. Therefore, it is likely that the majority of the 896,196 variants defined as the V1a2 -KO strain-specific originated either from the parental fish prior to genome-editing or accumulated during successive generations under captive breeding. This highlights the importance of sampling and preserving the genomic information of the founder or parental individuals when conducting off-target analysis in genome-edited organisms, which was unfortunately missing in our case. Given the large number of variants (896,196) and mutagenesis candidate sites (1,000), the possibility of overlapping by chance cannot be excluded. To assess this possibility, we calculated the expected number of overlaps using the following formula. The polymorphism density specific to the V1a2 -KO strain on the chub mackerel genome is denoted as \(\:{D}_{P}\) (variants/bp), and the total number of strain-specific variants (assumed to be SNPs) is \(\:{V}_{KO}=\:\text{896,196}\) . The whole-genome-length ( \(\:WGL\) ) is 828,681,152 bp. Based on this, the expected number of variants ( \(\:{E}_{17.5}\) ) within 1,000 randomly selected sequences of 17.5 bp in average length is calculated as: $$\:{E}_{17.5}=\:17.5\times\:1000\times\:\frac{{V}_{KO}}{WGL}\:\approx\:18.9$$ In practice, since repetitive elements and unannotated regions are often excluded from analyses, the effective genome size is smaller, and the actual variant density would be higher, increasing the expected overlap. These findings suggest that the 23 overlapping sites (excluding the on-target site) are most likely the result of spontaneous mutations that occurred either prior to TALEN treatment or during maintenance of the strain, rather than being caused by off-target activity. This interpretation is further supported by differences in binding scores and mismatch numbers between the on-target and off-target candidate sites (Supplementary data). Collectively, we found no evidence of off-target mutations were introduced in the V1a2 -KO strain. Although this study focused only on sequence homology, detailed verification using docking simulation through (AlphaFold3) [48], and other methods will be necessary to understand actual physiological phenomena. Conclusion In this analysis, a genome-wide off-target assessment was performed by a simple workflow that compared mutations across predicted TALEN binding sites of genome-edited individuals with their close and distant relatives and no exonic variants were detected. The result of this study strongly raises the possibility of aquaculture of the V1a2 -KO chub mackerel strain in future. While the methodology is expected to be an invaluable tool for design of fish genome-editing program and off target analysis. However, future investigations are pertinent to narrow down the consequences of domestication and other effects and unveil the physiological consequences of these mutations in the progeny as well as on consumer health. Abbreviations V1a2 : arginine vasotocin receptor V1a2 TALEN: Transcription Activator-Like Effector Nuclease KO: knockout SNP: Single Nucleotide Polymorphism INDEL: Insertion/Deletion DSB: Double Strand Break CDS: coding sequence Declarations Ethics approval and consent to participate All experimental protocols here in used were approved by the Institutional Animal Care and Use Committee of Kyushu University. Our manuscript confirms that the study has been reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Consent for publication The authors are aware that BMC Biotechnology is an open access journal that levies an article processing charge per article accepted for publication. The authors understand and agree to the open access policy. Availability of data and materials The genome sequence (GCF_027409825.1_fScoJap1.pri_genomic.fna.gz) and annotation file (GCF_027409825.1_fScoJap1.pri_genomic.gtf.gz) used for the analyses are publicly available from the National Center for Biotechnology Information (NCBI) at the following URL: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/ VCF files containing variant information for each sample are available from the corresponding author upon reasonable request. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was supported by the Research and implementation promotion program through open innovation grants（JPJ011937） from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN), JST SPRING, grant number JPMJSP2136, G7 grant foundation, Japan, and JSPS KAKENHI grant numbers JP15H02459, JP22K05832. Authors' contributions R.T., K.O. and T.S. analyzed and wrote the main manuscript text. H.O., N.N. and T.I. established the KO-strain, T.C., K.O. and M.M. correct the main manuscript text. Acknowledgements The authors would like to express appreciation to Dr. Yamamoto from the Graduate School of Integrated Sciences for Life, Hiroshima University, and Dr. Fujiwara from the Japan Fisheries research and Education Agency for their valuable contributions to the establishment of the KO strain. 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TALEN‐mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield. Plant biotechnology journal , 16 (4), 856-866. Becker, S., & Boch, J. (2021). TALE and TALEN genome editing technologies. Gene and Genome Editing , 2 , 100007. Barrett, S. C. H., & Charlesworth, D. (1991). Effects of a change in the level of inbreeding on the genetic load. Nature, 352 (6335), 522-524. Charlesworth, D., & Willis, J. H. (2009). The genetics of inbreeding depression. Nature reviews genetics, 10 (11), 783-796. Nishijima, W., Umehara, A., Okuda, T., & Nakai, S. (2015). Variations in macrobenthic community structures in relation to environmental variables in the Seto Inland Sea, Japan. Marine Pollution Bulletin , 92 (1-2), 90-98. Tang, H., Mao, S., Xu, X., Li, J., & Shen, Y. (2024). Genetic diversity analysis of different geographic populations of black carp (Mylopharyngodon piceus) based on whole genome SNP markers. Aquaculture, 582 , 740542. Buso, P., Diblasi, C., Manousi, D., Kwak, J. S., Vera-Ponce de Leon, A., Stenløkk, K., ... & Saitou, M. (2025). Parallel Selection in Domesticated Atlantic Salmon from Divergent Founders Including on Whole-Genome Duplication-derived Homeologous Regions. Genome Biology and Evolution , 17 (4), evaf063. Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., ... & Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature , 630 (8016), 493-500. Tables Table 2 and 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tablelist.xlsx analysisdataset.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 Oct, 2025 Reviewers agreed at journal 22 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor assigned by journal 02 Sep, 2025 Editor invited by journal 07 Aug, 2025 Submission checks completed at journal 04 Aug, 2025 First submitted to journal 04 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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. 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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-7219894","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516078923,"identity":"340914ee-aa7d-43fd-b557-d54a2b00e1d8","order_by":0,"name":"Ryo Takeuchi","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Takeuchi","suffix":""},{"id":516078924,"identity":"dc23f9ca-4d74-46e8-8967-4cf01167d232","order_by":1,"name":"Kaoru Ohno","email":"","orcid":"","institution":"National Institute for Basic 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1","display":"","copyAsset":false,"role":"figure","size":83632,"visible":true,"origin":"","legend":"\u003cp\u003eVisualization of target sites by iGV. The target region, ranging from 15,732,960 bp to 15,733,062 bp of chromosome 5, of four\u003cem\u003eV1a2\u003c/em\u003e-KO strain samples are compared with two Q-saba pools. The region, marked with red square was detected by GATK4 as a 13-base deletion only in the \u003cem\u003eV1a2\u003c/em\u003e-KO strain.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7219894/v1/9cc45b8e2a5d3bef4dd2d07b.png"},{"id":91852742,"identity":"71989b5c-6913-4590-8053-8a04ebf517b6","added_by":"auto","created_at":"2025-09-22 11:38:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177818,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of detected variants and candidate sites for mutagenesis. 3,959,178 common variants were detected in \u003cem\u003eV1a2\u003c/em\u003e-KO strain samples, and 896,196 variants specific to the \u003cem\u003eV1a2\u003c/em\u003e-KO strain were found in these variants compared to the Q-Saba samples. Comparison of 896,196 variants with 1,000 candidate sites for mutagenesis revealed 24 duplications (one of which was \u003cem\u003eV1a2\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7219894/v1/45cfde78c20ee7b5f3b9c0f9.png"},{"id":91855142,"identity":"6cd88544-d550-4287-88b6-6794e58cc0ac","added_by":"auto","created_at":"2025-09-22 11:54:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":706306,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7219894/v1/1f84e7be-b567-41cc-9d46-ac88bd0d3f4c.pdf"},{"id":91852748,"identity":"1f524e95-b5cc-4c46-8b85-19f71a3cb351","added_by":"auto","created_at":"2025-09-22 11:38:15","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16165,"visible":true,"origin":"","legend":"","description":"","filename":"Tablelist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7219894/v1/0ced45416629cf9b2af0d75e.xlsx"},{"id":91852747,"identity":"d849e8d2-2f99-4295-86a0-ac14426e3d63","added_by":"auto","created_at":"2025-09-22 11:38:15","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23118417,"visible":true,"origin":"","legend":"","description":"","filename":"analysisdataset.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7219894/v1/042980ddb7f079570aec7bfd.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide off-target assessment on the arginine vasotocin receptor V1a2 knockout Scomber japonicus (chub mackerel)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrowing population and its associated food demand are expected to create a protein crisis and according to food and agriculture organization of the United Nations (FAO) fish culture especially aquaculture of improved varieties are potentially feasible way out [1]. On the other hand, capture fisheries (i.e., fish caught directly from the wild) have become stagnant, showing no signs of recovery [2], thereby further emphasizing the need for sustainable aquaculture of suitable fish strains and innovative fish breeding technologies. Generally, selective breeding is the most practiced method to elevate the performance and strain production, but recent advances in genome-editing technology made them a formidable choice to develop strains of desirable traits [3, 4, 5, 6]. While selective breeding requires a long time and considerable financial resources, genome-editing demands-controlled laboratory environments, comprehensive genomic data, and advanced technical expertise. Over the past decades, genome-editing tools such as ZFN, TALEN, and CRISPR/Cas systems which induce double strand breaks (DSBs) in genomic DNA to generate functional mutations (knockout, KO) [7, 8, 9, 10] have emerged as widely preferred and alternative technologies for sustainable food production. These genome-editing tools have contributed greatly to solving genetic diseases, analyzing gene function across crop plants and farmed animals [11, 12] and improve productivity. The success of genome-edited products has even encourages policy makers to rethink and even legalize genome-edited crops and fishes for mass propagation and consumptions [13]. For instance, \u003cem\u003emyostatin\u003c/em\u003e-KO red sea bream [14] and a GABA-enriched tomato [15] are being approved for mass production and consumption in Japan.\u003c/p\u003e\n\u003cp\u003eWhile genome-editing tools are attracting attention as a next-generation aquatic breeding system, one concern is the introduction of unwanted mutations (off-target mutations) by genome-editing tools [16, 17, 18, 19]. Genome-editing tools can be engineered to make extremely well-defined alterations to the intended target genomic locus, but one potential complication is that the engineered nuclease will create other, various degree of unintended genomic changes i.e. \u0026ldquo;off-target\u0026rdquo; activity due to imperfect specificity. Proof of the absence of genome-wide off-target mutations is necessary to dispel psychological concerns [20, 21].\u003c/p\u003e\n\u003cp\u003eRecently, our research team has also developed a docile chub mackerel with reduced cannibalism by mutating of \u003cem\u003earginine vasotocin receptor V1a2\u003c/em\u003e (hereafter referred as \u003cem\u003eV1a2\u003c/em\u003e- KO) by TALEN in the \u003cem\u003eScomber japonicus\u003c/em\u003e (chub mackerel), a marine aquaculture model [22]. Apart from low aggression and reduced cannibalism, this knockout strain does not exhibit any other phenotypic differences compared to wild counterparts and thus expected to greatly contribute chub mackerel seed production. As a common marine fish inhabiting temperate and subtropical waters, genome-edited mackerel represents a promising candidate for enhancing the sustainability of marine aquaculture. Therefore, elucidating off-target effects is of paramount importance to ensure the confidence of both producers and consumers. In this study, to ascertain the safety issue, and evaluate the presence of unwanted mutation or genome-integration, we performed genome-wide off-target prediction and assessment to confirm whether serious mutations occur in \u003cem\u003eV1a2\u003c/em\u003e-KO chub mackerel produced by genome-editing.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003e\u003cu\u003eExperimental Fish maintenance:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAll of the chub mackerel used in the experiment were raised in the P1A marine aquarium facility of ABRIC Karatsu Satellite of Kyushu University. The\u003cem\u003eV1a2\u003c/em\u003e-KO strain chub mackerel was knocked out as shown in our previous study [22]. F0 generation of\u003cem\u003e \u003c/em\u003ethe \u003cem\u003eV1a2\u003c/em\u003e-KO strain chub mackerel was produced in 2017, F1 heterozygous progenies were prepared by crossing the potential F0 adult mutant males with chub mackerel females purchased from aquaculture firm in Oita (Japan), while the F2 and F3 were produced by crossing heterozygous \u003cem\u003eV1a2\u003c/em\u003e mutants the genome was extracted from the F3 generation \u003cem\u003eV1a2\u003c/em\u003e-KO strain chub mackerel were used in this study. Our own chub mackerel strain (Q-saba: https://www.city.karatsu.lg.jp/page/1431.html) were reared in the same facility and F7 generation was used in this investigation. Fish breeding and rearing were performed using previously published protocols [22, 23]. \u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDNA library preparation and analysis:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eHigh quality DNA were collected from gills. using Qiagen blood and tissue DNA isolation kit following manufacturer\u0026rsquo;s instructions. DNA libraries were prepared using standard Illumina DNA library preparation protocol and sequenced using Illumina short read sequencing (Table. 2). 9 sample groups covering a total of 4 \u003cem\u003eV1a2-\u003c/em\u003eKO homozygous fish (2/ sex) and 49 cultured strains (Q-saba) (Table.1). \u003c/p\u003e\n\u003cp\u003eTable 1. Sample name list.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eDetails\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cem\u003eV1a2-\u003c/em\u003eKO-F1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eKO strain F3 generation Female, 1 sample\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cem\u003eV1a2\u003c/em\u003e-KO-F5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eKO strain F3 generation Female, 1 sample\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cem\u003eV1a2\u003c/em\u003e-KO-M1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eKO strain F3 generation Male, 1 sample\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003e\u003cem\u003eV1a2\u003c/em\u003e-KO-M4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eKO strain F3 generation Male, 1 sample\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eQsaba-Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eFarmed chub mackerel female, for genome-estimation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eQsaba-F-pool\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eFarmed chub mackerel female group, 12 samples pool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eQsaba-M-pool\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eFarmed chub mackerel male group, 12 samples pool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eQsaba-L-pool\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eFarmed chub mackerel large group, 12 samples pool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 139px;\"\u003e\n \u003cp\u003eQsaba-S-pool\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 436px;\"\u003e\n \u003cp\u003eFarmed chub mackerel small group, 12 samples pool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cu\u003eEstimation of TALEN-mutagenesis sites:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTALEN induces DSB by preparing two sets of complexes containing a nuclease (FokI) (Fig 2). The optimal theoretical conditions for TALEN-based DNA editing are that the TALEN dimer must bind to the genomic DNA and that the spacer sequence should be 12-24 bases in length [24]. The TALEN pair (left, 5ʹ-TTCCGCTTCTATGGTCCA-3ʹ and right, 5ʹ-TGGAGGTGTTTGACTAT-3ʹ), having a spacer of 14 bp, was used to target the S\u0026ndash;S binding site of \u003cem\u003eV1a2\u003c/em\u003e and create the KO strain. In this study, potential TALEN mutagenesis sites were identified using Paired Target Finder (https://tale-nt.cac.cornell.edu/node/add/talef-off-paired) [25] in accordance with the previous reports [26, 27]. The GCF_027409825.1_fScoJap1.pri_genomic.fna.gz (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/) were as used as the reference genome database [28]. The RVD sequences were provided as follows; RVD sequence 1, NG HD HD NN HD NG NG HD NG NI NG NN NN NG HD HD NI NG; RVD sequence 2, NN NN NI NN NN NG NN NG NG NG NN NI HD NG NI NG. Minimum and maximum spacer lengths were set to 10 and 25, respectively. Other parameters were set as recommended. From the list of candidate mutagenesis sites, the top 1,000 sites with the lowest combined TAL1 and TAL2 scores were selected for downstream analysis, as these were expected to represent the highest binding specificity. \u003c/p\u003e\n\u003cp\u003e\u003cu\u003eRaw reads processing and variant calling:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe raw read data were processed by fastp (v0.24.0) [29] and mapped to the chub mackerel genome (NCBI) as a reference genome by snap-aligner (v2.0.3) [30]. Low-quality reads were removed by ngsutils (v0.5.9, -mismatch 4 -properpair) [31]. Tentative variant calls and SNPs/INDELs filtering were then performed by GATK4 [32]. Variant calls by GATK4 (v4.6.1) were performed on all samples. The resulting SNPs/INDELs were treated as known sites and mutations were extracted by GATK4 once again (https://gatk.broadinstitute.org/hc/en-us/sections/360007226651-Best-Practices-Workflows). The bam file created was visualized by iGV (v2.18.2) [33], whenever necessary. \u003c/p\u003e\n\u003cp\u003e\u003cu\u003eExtraction of common and specific mutations in the \u003cem\u003eV1a2\u003c/em\u003e-KO strain chub mackerel:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAfter variants calling, the resulting vcf file and bcftools (v1.21) [34] were used to extract variants common to the \u003cem\u003eV1a2\u003c/em\u003e-KO strain. Additional filtering was performed by removing the variants common to both \u003cem\u003eV1a2\u003c/em\u003e and Q-saba samples. Common variants were compared to DSB candidate sites (1,000 sites) by TALEN. The sites where the location of these variants overlapped with DSB candidate sites were treated as potential off-target-mutations sites. Further, the position of the mutation was compared to the position of the spacer sequence, and if the mutation existed within the spacer sequence, it was evaluated as a mutation that could have been caused by TALEN. \u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePhysiological assessment of off target effects:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe potential off target sites were evaluated by aligning the individual sites to annotation database of chub mackerel (gtf format file: https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/GCF_027409825.1_fScoJap1.pri_genomic.gtf.gz). The annotated genes, if any, were identified and evaluated further. Particularly, occurrence of frameshift mutation, changes in amino acid sequences and intronic and exonic mutation (changing the overall gene structure) were carefully checked.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cu\u003eTALEN binding site listing\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eGenome-editing through TALEN, CRISPR/Cas9 or other techniques creates DSB, but their activity and feasibility depends on the quantitative measurement of both on-target and off-target activity. Unlike the pseudo-random nature of retro or lenti-viral integration sites, nuclease off-target activity is presumed to be a direct result of the nuclease binding a nearby sequence with some level of homology to the intended target, and subsequently inducing a DSB. The elements that define the specificity of TALEN are the Left/Right TALEN motif sequence, DNA binding strength, and spacer length (Fig. 2). In this analysis, these were parameterized and adjusted to estimate the sites where DSB may have occurred.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the top 1,000 candidate sites with the lowest binding scores, the site with the highest predicted binding affinity (i.e., the lowest total score), corresponded to the intended target: NC_070582.1, TAL1: 15,732,984; TAL2: 15,733,031, with a total score of 12.76. The second-ranked site was located at NC_070583.1, TAL1: 6,575,345; TAL2: 6,575,398, with a total score of 26.5, which is more than twice the score of the target site. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eOff-target evaluation by \u003cem\u003eV1a2\u003c/em\u003e-KO strain-specific variants and TALEN binding sites\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eWhole-genome resequencing using Illumina short-read seq produced between 137,905,856 and 780,774,690 reads, of which 67,232,147 to 458,545,005 reads were successfully mapped to the chub mackerel reference genome (Table\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003eVariant calling using GATK4 for the four\u003cem\u003e\u0026nbsp;V1a2\u003c/em\u003e-KO strains identified approximately 7.0 to 7.7 million variants per sample, corresponding to a variation rate of 9.7\u0026ndash;11.0%. In comparison, the control Q-saba samples exhibited 2.5\u0026ndash;11.3% variation, with a total of 8,258,318 to 11,528,230 variants (Table 2). However, only 3,959,178 variants were found to be common across all four\u003cem\u003e\u0026nbsp;V1a2\u003c/em\u003e-KO individuals. Importantly, a 13-base deletion in the \u003cem\u003eV1a2\u003c/em\u003e, the intended genome-editing target, was consistently detected in all four \u003cem\u003eV1a2\u003c/em\u003e-KO individuals (Fig. 1).\u003c/p\u003e\n\u003cp\u003eNotably, both the \u003cem\u003eV1a2\u003c/em\u003e-KO and Q-saba strains used in this study originated from Oita, Japan, whereas the reference genome was established from a Korean population. Although chub mackerel is a highly migratory species, genetic polymorphisms arising from geographic isolation cannot be completely ruled out. To address this, we compared the NGS variant profiles of the\u003cem\u003e\u0026nbsp;V1a2\u003c/em\u003e-KO and Q-saba strains maintained under identical laboratory conditions in Saga, Japan.\u003c/p\u003e\n\u003cp\u003eOf the 3,959,178 shared variants observed in the \u003cem\u003eV1a2\u003c/em\u003e-KO strain, 896,196 variants (22.64%) were unique to this strain and not detected in Q-saba. The remaining variants were shared between the two groups (Table 1). To assess whether these \u003cem\u003eV1a2\u003c/em\u003e-KO-specific variants may be attributable to TALEN-mediated off-target effects, we compared them against the previously identified top 1,000 mutagenesis candidate sites. This comparison revealed 24 overlapping sites, including the intended \u003cem\u003eV1a2\u0026nbsp;\u003c/em\u003etarget gene site. For the remaining 23 overlapping sites (excluding the intended \u003cem\u003eV1a2\u003c/em\u003e target) comparison with publicly available gene annotations revealed no overlap with CDSs or intragenic introns (Table 3, Supplementary data). In other words, no evidence of TALEN-induced disruptions in protein-coding regions was found, and no unintended protein synthesis abnormalities were inferred outside of the \u003cem\u003eV1a2\u003c/em\u003e gene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Details of mutations in each sample\u003c/p\u003e\n\u003cp\u003e(View Tables file)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Details of 23 off-target sites and \u003cem\u003eV1a2\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(View Tables file)\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eProper evaluation of genome-edited food products for off-target effects are of utmost importance to assess the consequences of genome-editing on both species physiology and consumer health.\u003c/p\u003e\u003cp\u003eIn this study, we conducted the first comprehensive evaluation of genome-editing effects in a marine teleost fish by using whole-genome sequencing data from the F3 generation of the\u003cem\u003eV1a2\u003c/em\u003e-KO chub mackerel. We first identified 1,000 potential mutagenesis candidate sites across the genome based on TALEN binding predictions, and then detected 24 strain-specific variants present only in the \u003cem\u003eV1a2\u003c/em\u003e-KO lineage. Finally, we assessed whether these 24 variants were caused by TALEN-mediated genome-editing or were spontaneous in origin.\u003c/p\u003e\u003cp\u003eTALEN is widely used for genome-editing in plants to humans and infect [35], the first genome-edited crop, genome-edited soybean [36], that has reached the market was also generated using TALEN. TALEN requires a longer sequence length specification (30\u0026ndash;40 bp) compared to other well-known genome-editing tools such as ZFN (9\u0026ndash;18 bp) and CRISPR/Cas system (20 bp\u0026thinsp;+\u0026thinsp;PAM) [37, 38, 39] and thus, are predicted to be one of highly-specific genome-editing tools. We have used TALEN mRNA for the development of the \u003cem\u003eV1a2\u003c/em\u003e mutant and successfully obtained a specific mutant line with 13 base deletion [22]. Unlike previous reports [40], so far, we were unable to identify any genomic integration of reverse transcribed TALEN mRNAs highlighting the possibility of commercialization of this product. In plants, TALEN has been successfully employed to efficiently modify multiple gene copies simultaneously, for example, 107 out of 109 copies and alleles of the \u003cem\u003ecaffeic acid O-methyltransferase\u003c/em\u003e (\u003cem\u003eCOMT\u003c/em\u003e) gene in sugarcane were edited without any visible phenotypic defects [41, 42]. Notably, fish genomes have undergone whole-genome duplication during evolution and often contain two to four copies of the same gene. These characteristic makes TALEN a promising tool for generating food-grade, off-target-free genome-edited fish in a relatively short time. However, in present investigation, only a single TALEN pair targeting a single gene copy was used, and only one mutant line was analyzed. This limited scope restricts our ability to fully assess nuclease binding efficiency, the most reliable prediction methods for target site selection, and species-specific genome-editing dynamics and thus warrants further investigations evaluate the broader applicability of TALEN in fish species.\u003c/p\u003e\u003cp\u003eGenetic mutations are often overrepresented in the inbreed strains [43, 44] and thus off-target assessment should consider comparing parents and progeny, wild and cultured strains, and geographically isolated population. In the studied \u003cem\u003eV1a2\u003c/em\u003e-KO strain, 3,062,982 out of 3,959,178 variants (77.4%) were found to be shared with the Q-saba strain. This high degree of overlap suggests substantial genetic diversity between the reference chub mackerel genome (derived from a Korean population) and the Oita-derived strains (\u003cem\u003eV1a2\u003c/em\u003e-KO and Q-saba) used in this study. The observed differences may reflect underlying population effects, as the reference genome originated from South Korea [26], whereas both the \u003cem\u003eV1a2\u003c/em\u003e-KO and Q-saba strains were developed from fish collected in Oita, Japan. Geographic separation can lead to genetic diversification between populations, including the formation of distinct haplotypes, SNPs and INDELs [45, 46]. Whether such inter-population genetic differences can influence genome-editing efficiency or outcomes remains unclear due to limited available data and require further investigation in future. Additionally, genetic mutations may have been accumulated during domestication and selective breeding processes [47]. Therefore, it is likely that the majority of the 896,196 variants defined as the \u003cem\u003eV1a2\u003c/em\u003e-KO strain-specific originated either from the parental fish prior to genome-editing or accumulated during successive generations under captive breeding. This highlights the importance of sampling and preserving the genomic information of the founder or parental individuals when conducting off-target analysis in genome-edited organisms, which was unfortunately missing in our case.\u003c/p\u003e\u003cp\u003eGiven the large number of variants (896,196) and mutagenesis candidate sites (1,000), the possibility of overlapping by chance cannot be excluded. To assess this possibility, we calculated the expected number of overlaps using the following formula. The polymorphism density specific to the \u003cem\u003eV1a2\u003c/em\u003e-KO strain on the chub mackerel genome is denoted as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{D}_{P}\\)\u003c/span\u003e\u003c/span\u003e(variants/bp), and the total number of strain-specific variants (assumed to be SNPs) is \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{KO}=\\:\\text{896,196}\\)\u003c/span\u003e\u003c/span\u003e. The whole-genome-length (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:WGL\\)\u003c/span\u003e\u003c/span\u003e) is 828,681,152 bp. Based on this, the expected number of variants (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{17.5}\\)\u003c/span\u003e\u003c/span\u003e) within 1,000 randomly selected sequences of 17.5 bp in average length is calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{E}_{17.5}=\\:17.5\\times\\:1000\\times\\:\\frac{{V}_{KO}}{WGL}\\:\\approx\\:18.9$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn practice, since repetitive elements and unannotated regions are often excluded from analyses, the effective genome size is smaller, and the actual variant density would be higher, increasing the expected overlap. These findings suggest that the 23 overlapping sites (excluding the on-target site) are most likely the result of spontaneous mutations that occurred either prior to TALEN treatment or during maintenance of the strain, rather than being caused by off-target activity. This interpretation is further supported by differences in binding scores and mismatch numbers between the on-target and off-target candidate sites (Supplementary data). Collectively, we found no evidence of off-target mutations were introduced in the \u003cem\u003eV1a2\u003c/em\u003e-KO strain. Although this study focused only on sequence homology, detailed verification using docking simulation through (AlphaFold3) [48], and other methods will be necessary to understand actual physiological phenomena.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this analysis, a genome-wide off-target assessment was performed by a simple workflow that compared mutations across predicted TALEN binding sites of genome-edited individuals with their close and distant relatives and no exonic variants were detected.\u003c/p\u003e\u003cp\u003eThe result of this study strongly raises the possibility of aquaculture of the\u003cem\u003eV1a2\u003c/em\u003e-KO chub mackerel strain in future. While the methodology is expected to be an invaluable tool for design of fish genome-editing program and off target analysis.\u003c/p\u003e\u003cp\u003eHowever, future investigations are pertinent to narrow down the consequences of domestication and other effects and unveil the physiological consequences of these mutations in the progeny as well as on consumer health.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eV1a2\u003c/em\u003e: \u003cem\u003earginine vasotocin receptor V1a2\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTALEN: Transcription Activator-Like Effector Nuclease\u003c/p\u003e\n\u003cp\u003eKO: knockout\u003c/p\u003e\n\u003cp\u003eSNP: Single Nucleotide Polymorphism\u003c/p\u003e\n\u003cp\u003eINDEL: Insertion/Deletion\u003c/p\u003e\n\u003cp\u003eDSB: Double Strand Break\u003c/p\u003e\n\u003cp\u003eCDS: coding sequence\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll experimental protocols here in used were approved by the Institutional Animal Care and Use Committee of Kyushu University. Our manuscript confirms that the study has been reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eThe authors are aware that BMC Biotechnology is an open access journal that levies an article processing charge per article accepted for publication. The authors understand and agree to the open access policy.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe genome sequence (GCF_027409825.1_fScoJap1.pri_genomic.fna.gz) and annotation file (GCF_027409825.1_fScoJap1.pri_genomic.gtf.gz) used for the analyses are publicly available from the National Center for Biotechnology Information (NCBI) at the following URL:\u003cbr\u003e\u0026nbsp;https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/409/825/GCF_027409825.1_fScoJap1.pri/\u003cbr\u003e\u0026nbsp;VCF files containing variant information for each sample are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Research and implementation promotion program through open innovation grants（JPJ011937） from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN), JST SPRING, grant number JPMJSP2136, G7 grant foundation, Japan, and JSPS KAKENHI grant numbers JP15H02459, JP22K05832.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eR.T., K.O. and T.S. analyzed and wrote the main manuscript text. H.O., N.N. and T.I. established the KO-strain, T.C., K.O. and M.M. correct the main manuscript text.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors would like to express appreciation to Dr. Yamamoto from the Graduate School of Integrated Sciences for Life, Hiroshima University, and Dr. Fujiwara from the Japan Fisheries research and Education Agency for their valuable contributions to the establishment of the KO strain.\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to all the staff at the Aqua-Bioresource Innovation Center (ABRIC) Karatsu Satellite, Kyushu University, and Karatsu Fisheries Revitalization Support Center (Saga, Japan) for their assistance with fish maintenance and sampling support. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWeis, T. (2013). 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Accurate structure prediction of biomolecular interactions with AlphaFold 3. \u003cem\u003eNature\u003c/em\u003e, \u003cem\u003e630\u003c/em\u003e(8016), 493-500.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 2 and 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chub mackerel, TALEN, Off-target mutation, Double strand break, Sequence homology","lastPublishedDoi":"10.21203/rs.3.rs-7219894/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7219894/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIn recent decades, genome-edited fish production is gaining momentum among aquaculturists, but uncertainties about off-target mutations is a major concern for commercialization of genome-edited fish products. Recently, a knockout (KO) \u003cem\u003eScomber japonicus\u003c/em\u003e (chub mackerel) strain of \u003cem\u003earginine vasotocin receptor V1a2\u003c/em\u003e (\u003cem\u003eV1a2\u003c/em\u003e) was developed using TALEN to reduce the cannibalism frequency of fry. Aside from this behavioral trait, the KO fish had no other phenotypic differences with their wild counterpart. Whether the changed trait is an outcome of unwanted mutation in the genome or not, is a prerequisite for consumer\u0026rsquo;s safety and psychological satisfaction and thus needs careful genome-wide analysis. Therefore, the objective of this study was to detect and evaluate potential off-target sites in the genome-edited \u003cem\u003eV1a2\u003c/em\u003e-KO chub mackerel strain.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn the \u003cem\u003eV1a2\u003c/em\u003e-KO strain, approximately 0.9\u0026nbsp;million variants were identified through specific variant extraction. To assess potential off-target effects, putative mutagenesis sites for TALEN were predicted using the chub mackerel reference genome (available at NCBI), and the top 1,000 sites with lowest binding scores (indicating stronger predicted binding affinity) were selected. Comparison of these 1,000 predicted TALEN binding sites with ~\u0026thinsp;0.9\u0026nbsp;million variants revealed 24 overlapping sites. Among them, only one site, corresponding to the target gene \u003cem\u003eV1a2\u003c/em\u003e (13-base deletion), having the lowest binding score of 12. 76, was located within a coding sequence (CDS), suggesting no phenotypic side effects in the \u003cem\u003eV1a2\u003c/em\u003e-KO strain. Additional, expected number of variants analysis confirmed that the remaining observed variations might be an outcome of spontaneous mutation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eUsing genome-wide assessment and comparison with predicted TALEN binding sites, of genome-edited individuals with their close and distant relatives, we have identified 24 probable sites, among which only one, the 13 base deletion was located in the target gene\u0026rsquo;s CDS strongly indicating no off-target associated phenotypic changes. Cumulatively, our results confirms the precision of TALEN genome-editing system and safety of TALEN genome-edited products and strongly raises the possibility of commercialization of \u003cem\u003eV1a2\u003c/em\u003e-KO chub mackerel strain in future.\u003c/p\u003e","manuscriptTitle":"Genome-wide off-target assessment on the arginine vasotocin receptor V1a2 knockout Scomber japonicus (chub mackerel)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 11:38:09","doi":"10.21203/rs.3.rs-7219894/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-10-14T20:10:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87506538130442573537343925414863158229","date":"2025-09-22T16:41:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-02T04:22:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T04:07:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-07T07:28:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-05T01:38:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biotechnology","date":"2025-08-05T01:35:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d449777-5644-4fda-b264-f1564c79e4f3","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T11:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 11:38:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7219894","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7219894","identity":"rs-7219894","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}