Evaluation method for genetic stability of target traits of genetically edited sheep

Abstract

The invention provides a genetic stability evaluation method for target traits of gene editing sheep, and relates to the technical field of gene editing and animal genetic breeding. According to the method, F0 to F3 generation gene editing sheep populations are constructed, a target gene is accurately edited in combination with a CRISPR / Cas9 technology, and individuals with high editing efficiency, low chimeric degree and low off-target risk are strictly screened in the F0 generation; analyzing genotype separation rules and phenotype genetic consistency through multi-generation pedigree tracking; standardized phenotype determination is carried out in a plurality of growth stages, genotype, phenotype and off-target data are integrated, a genetic stability index quantitative model is established, and the stability can be calculated. The method realizes the crossing from qualitative judgment to quantitative evaluation, has the advantages of strong systematicness, data traceability, high safety and good universality, and provides reliable technical support for variety breeding and biological safety evaluation of the gene editing sheep.

Description

Technical Field

[0001] This invention relates to the fields of gene editing and animal genetic breeding technology, and more specifically, to a method for assessing the genetic stability of a target trait in gene-edited sheep. Background Technology

[0002] In recent years, with the rapid development of biotechnology, gene editing technology has shown great potential and application value in the field of livestock breeding. Among them, representative gene editing technologies such as CRISPR / Cas9, TALEN, and ZFN, with their precision and efficiency, have successfully achieved targeted improvement of several important target traits in sheep.

[0003] Regarding growth rate, researchers have used gene editing technology to regulate genes related to growth and development, enabling sheep to grow and gain weight more rapidly. This not only shortens the breeding cycle and improves breeding efficiency but also increases the economic benefits for farmers. In terms of disease resistance, gene editing technology can modify immune-related genes in sheep, enhancing their resistance to common diseases, reducing the occurrence and spread of diseases, and lowering breeding costs and risks. For wool quality improvement, researchers have edited genes related to wool growth and quality, making sheep wool finer, softer, and shinier, improving wool quality and market value. Regarding reproductive performance enhancement, gene editing technology can regulate reproductive genes in sheep, increasing the number of lambs born to ewes and improving reproductive efficiency, providing a strong guarantee for the sustainable development of the sheep industry.

[0004] Although gene editing technology has achieved remarkable results in sheep breeding, gene-edited individuals have revealed a series of problems in subsequent reproduction, which seriously restricts the promotion and application of this technology in actual breeding.

[0005] Unstable expression of the target trait is a common problem. In the offspring of gene-edited sheep, the originally edited target trait may not be expressed stably as expected. For example, sheep whose growth rate has been improved through gene editing may have offspring with slower or fluctuating growth rates, making the improvement in the target trait unsustainable. Off-target effects are also a significant concern. Although gene editing technology has high precision, there is still a certain probability of off-target effects, meaning that the editing process may affect non-target genes. These off-target effects may lead to unexpected trait changes or health problems in sheep, affecting their normal growth and reproduction. Gene silencing also occurs frequently, where the edited gene is suppressed during subsequent expression and cannot function properly, preventing the target trait from being expressed. In addition, genetic segregation is also quite common. In the offspring of gene-edited sheep, the target trait may segregate, with some offspring expressing the target trait while others do not, resulting in the target trait not being stably inherited by offspring.

[0006] Currently, the assessment of genetic stability in gene-edited animals relies heavily on traditional phenotypic observation and limited-generation genotyping, which has significant limitations. While phenotypic observation is intuitive, it is subjective and biased. Different observers may interpret the same phenotype differently, and phenotypes are easily influenced by environmental factors, failing to accurately reflect the genetic essence of the target trait in gene-edited animals. Limited-generation genotyping cannot comprehensively understand the genetic changes of the target trait across multiple generations. Since the genetic stability of gene-edited animals requires long-term observation and analysis over multiple generations, existing genotyping often only covers a few generations, potentially missing important genetic information. Furthermore, existing methods have shortcomings in assessment duration, data integration, and analysis of genetic patterns. An overly short assessment period may fail to detect problems arising in the long-term reproduction of the target trait, leading to inaccurate results. Regarding data integration, existing methods often focus only on single types of data, such as genotype or phenotypic data, lacking integrated analysis of multi-dimensional data and failing to comprehensively reflect the genetic stability of the target trait. In analyzing genetic patterns, existing methods, due to limited data and insufficient analytical tools, are insufficient to deeply reveal the genetic laws governing the target traits of gene-edited sheep, thus failing to provide effective guidance for subsequent breeding work. Therefore, this paper proposes a method for assessing the genetic stability of the target traits in gene-edited sheep. Summary of the Invention

[0007] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a method for assessing the genetic stability of target traits in gene-edited sheep, comprising the following specific steps: Step 1: Construction of gene-editing vector and acquisition of F0 generation individuals. The design specifically targets functional genes in the sheep genome associated with the target trait, namely the MSTN myostatin gene (GenBank accession number NC_019467.1), exon 1 region, PRNP prion protein gene, and genes related to anti-pruritus or FGF5 hair follicle development regulation. The sgRNA sequence is 20 bp in length and has a 5'-N... 18-22 The GG-3' structure was confirmed by BLAST alignment to have unique targeting in the sheep genome, with an off-target prediction score of ≤3 using tools such as COSMID or CHOPCHOP. The sgRNA and Cas9 mRNA or Cas9 protein complex were introduced into the pronucleus of single-cell fertilized eggs of New Zealand white sheep or Hu sheep via microinjection. The injection concentrations were: Cas9 mRNA 50 ng / μL, sgRNA 25 ng / μL, and the injection volume was 10 pL / egg. Alternatively, the somatic cell nuclear transfer (SCNT) method can be used: CRISPR / Cas9-edited sheep ear fibroblasts are used as donor cells and transplanted into enucleated MII stage oocytes. The fusion voltage is 1.2 kV / cm, the pulse duration is 30 μs, and activation is performed by ionomycin 5 μmol / L for 5 min combined with 6-DMAP 2 mmol / L for 4 h. After the embryos are cultured in vitro to the morula or blastocyst stage, they are transferred into the uterus of a recipient ewe in estrus. During pregnancy, ultrasound monitoring confirms that the embryos are developing normally, and F0 generation lambs are born.

[0008] Step 2: Identification and Screening Criteria for F0 Generation Molecular Basis Genomic DNA was extracted from ear tissue of F0 generation individuals, and the target editing region was amplified by PCR. The amplification system was as follows: 100 ng template DNA, 0.4 μM each of forward and reverse primers, 200 μmol / L dNTPs, and 0.5 U high-fidelity Taq enzyme. The amplified products were initially screened by T7E1 enzyme digestion or high-resolution melting curve HRM. Positive samples were further subjected to TA cloning + Sanger sequencing or high-throughput sequencing amplicon-seq with a sequencing depth ≥1000×. Editing efficiency is defined as: the number of clones containing the edited allele in the test sample / the total number of clones × 100%, requiring an editing efficiency of ≥60% for a single F0 individual; Chimeras are classified as follows: if ≥2 different indel types are detected and the dominant type accounts for <80%, they are defined as highly chimeric and are not allowed to enter the breeding system; only low chimerism or quasi-homozygous F0 individuals with editing efficiency ≥60% and dominant edit type accounting for ≥80% are retained for subsequent breeding. Off-target detection uses whole-genome sequencing (WGS) with a sequencing depth ≥30 or targeted deep sequencing targeting the top 10 predicted off-target sites with a depth ≥1000. If the mutation frequency of any off-target site exceeds 0.5%, it is considered high-risk and the individual is excluded.

[0009] Step 3: Establishing a multi-generational pedigree Qualified F0 generation rams are mated with wild-type ewes of the same breed with the same genetic background and an inbreeding coefficient of <5% through natural mating or artificial insemination to obtain the F1 generation. Individuals carrying the target edited allele in the F1 generation were selected, confirmed by PCR and sequencing, and then crossed with siblings or half-sibs to obtain the F2 generation. In the F2 generation, homozygous edited individuals are selected and interbred to obtain the F3 generation; The number of individuals effectively involved in breeding in each generation shall not be less than 8, the sex ratio shall be close to 1:1, the inbreeding increment shall be controlled at <6.25% per generation to avoid full-sib mating, and the pedigree record shall include individual number, parent ID, date of birth, mating combination, genotype status, and record completeness rate ≥95%.

[0010] As a preferred technical solution of the present invention, the target trait is muscle growth trait. Phenotypic assessment is carried out at four stages: birth, 90 days after weaning, 6 months of age, and 12 months of age. The measurement indicators include weight, daily weight gain, height, length, and chest circumference. Among them, the increase in daily weight gain is considered to be not less than 15% in F1 generation heterozygotes and not less than 25% in F2 generation homozygotes.

[0011] As a preferred technical solution of the present invention, genotype detection is performed by PCR amplification combined with high-resolution melting curve analysis (HRM) or next-generation sequencing (NGS), the sequencing coverage depth of the target editing site is not less than 500×, and the genotype interpretation accuracy is ≥99%; the genetic transmission rate of the target allele in three consecutive generations of F1–F3 is not less than 85% of the theoretical value, and there are no reversion mutations or new insertion / deletion (indel) occurrences.

[0012] As a preferred embodiment of the present invention, the phenotypic stability score P is calculated according to the following formula: P=1-CV n / CV0 Among them, CV n CV0 is the phenotypic coefficient of variation for the target trait in the nth generation (n≥F1), and CV0 is the coefficient of variation for the wild-type control population. The P-value is normalized to the [0,1] interval. When the P-values ​​for F1–F3 generations are all ≥0.75, the phenotype is considered stable.

[0013] As a preferred embodiment of the present invention, the genotype consistency score G is determined in the following manner: G=N e / N t Where N e N represents the number of individuals in a generation that possess the expected target genotype, such as homozygous edited genotype. t The total number of individuals tested in this generation; a G value of ≥0.45 in the F2 generation meets the 1:2:1 segregation ratio, and a G value of ≥0.70 in the F3 generation is considered to indicate that the genotype is stabilizing.

[0014] As a preferred technical solution of the present invention, the genetic concordance score H is calculated by a chi-square test, using the following formula: H=1-(X 2 / X 2 0.05 ) Where X 2 X is the chi-square value of the actual genotype segregation ratio versus the theoretical Mendelian segregation ratio.2 0.05 The critical value is 5.99 when the degrees of freedom are 2; when H≥0.80, it is considered that the genetic law conforms to expectations.

[0015] As a preferred technical solution of the present invention, the genetic stability index (SSI) is calculated according to a weighted formula: SSI = 0.4 × P + 0.4 × G + 0.2 × H The weighting coefficients were determined by principal component analysis (PCA). When the average SSI of three consecutive generations F1–F3 is ≥0.80 and the lowest SSI of a single generation is ≥0.70, the target trait is considered to be genetically stable.

[0016] As a preferred technical solution of the present invention, the off-target effect assessment is carried out in the F0, F2 and F3 generations. GUIDE-seq or CIRCLE-seq is used to predict no less than 20 potential off-target sites, and their mutation frequency is verified by deep sequencing. If the mutation frequency of any off-target site in any generation exceeds 1.0%, it is determined to be genetically unstable and the propagation of the pedigree must be terminated.

[0017] As a preferred technical solution of the present invention, the environmental factor control includes: all individuals are raised in the same standardized sheepfold, with a temperature of 15–25℃, a relative humidity of 50–70%, and fed a uniform complete feed, with the average daily feed intake varying by no more than ±5%; phenotypic data collection is carried out by two or more technicians in a double-blind operation, and the measurement error is controlled within ±2%.

[0018] As a preferred technical solution of the present invention, a genetic evaluation database based on SQL or MongoDB is established to store pedigree information, genotype data, phenotypic records and environmental parameters, gene frequency dynamic curves and SSI heatmaps; the evaluation period is not less than 24 months, covering at least three reproductive cycles, and the data missing rate is less than 5% for final stability determination.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention establishes a continuous multi-generational pedigree tracing system and combines multi-dimensional data, including phenotypic data, genotypic data, transcriptomic and epigenetic information, to construct a comprehensive evaluation model. This cross-generational, multi-omics fusion analysis method effectively overcomes the one-sidedness and uncertainty of traditional evaluations that rely solely on single phenotypic observation or genotypic detection, significantly improving the accuracy of judging the genetic stability of target traits and ensuring that edited traits are truly and stably inherited in offspring. Through mathematical modeling, key parameters such as multi-generational phenotypic consistency, genotype transmission rate, phenotypic variation coefficient, and off-target frequency are weighted and integrated to generate a quantifiable comprehensive score.

[0020] The design of this invention is highly modular and configurable, applicable not only to the evaluation of various economic traits in sheep, such as growth, reproduction, disease resistance, and wool quality, but also to gene editing research in other livestock. Furthermore, this evaluation system is compatible with multiple gene editing technologies, including CRISPR / Cas9, Base Editing, and Prime Editing, and can flexibly adapt to the differences in genetic characteristics resulting from different editing strategies, demonstrating broad application prospects and promotional value.

[0021] By tracking the genetic behavior and unintended effects of edited genes across multiple generations over a long period, this invention can effectively identify potential risk factors such as genetic drift, chimera transmission, and off-target accumulation, providing systematic data support for assessing the long-term biosafety of gene-edited animals. This is of great significance for preventing gene pollution, ensuring ecological and environmental safety, and maintaining population genetic diversity.

[0022] The standardized evaluation process and quantitative indicators provided by this invention can serve as the technical basis for the approval of new gene-edited animal varieties, helping relevant authorities establish a scientific, transparent, and operable regulatory framework. Simultaneously, stable and reliable genetic performance data helps enhance the confidence of breeding companies, farmers, and consumers in gene-edited animal products, accelerating their industrialization and market promotion in the livestock industry. Attached Figure Description Figure 1 This is a screening diagram for the identification of F0 generation molecules provided by the present invention; Figure 2 A flowchart illustrating the process of establishing a multi-generation breeding pedigree provided by this invention; Figure 3 This is a data flow chart for genotype detection provided by the present invention; Figure 4 This is a data flowchart for the core assessment of genetic stability provided by the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are specific implementations of the present invention and are not limited to all embodiments.

[0024] Therefore, the following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0026] Example 1: A method for assessing the genetic stability of a target trait in gene-edited sheep, comprising the following specific steps: Step 1: Construction of gene-editing vector and acquisition of F0 generation individuals The design specifically targets functional genes in the sheep genome associated with the target trait, namely the MSTN myostatin gene (GenBank accession number NC_019467.1), exon 1 region, PRNP prion protein gene, and genes related to anti-pruritus or FGF5 hair follicle development regulation. The sgRNA sequence is 20 bp in length and has a 5'-N... 18-22 The GG-3' structure was confirmed by BLAST alignment to have unique targeting in the sheep genome, with an off-target prediction score of ≤3 using tools such as COSMID or CHOPCHOP. The sgRNA and Cas9 mRNA or Cas9 protein complex were introduced into the pronucleus of single-cell fertilized eggs of New Zealand white sheep or Hu sheep via microinjection. The injection concentrations were: Cas9 mRNA 50 ng / μL, sgRNA 25 ng / μL, and the injection volume was 10 pL / egg. Alternatively, the somatic cell nuclear transfer (SCNT) method can be used: CRISPR / Cas9-edited sheep ear fibroblasts are used as donor cells and transplanted into enucleated MII stage oocytes. The fusion voltage is 1.2 kV / cm, the pulse duration is 30 μs, and activation is performed by ionomycin 5 μmol / L for 5 min combined with 6-DMAP 2 mmol / L for 4 h. After the embryos are cultured in vitro to the morula or blastocyst stage, they are transferred into the uterus of a recipient ewe in estrus. During pregnancy, ultrasound monitoring confirms that the embryos are developing normally, and F0 generation lambs are born.

[0027] Step 2: Identification and Screening Criteria for F0 Generation Molecular Basis Genomic DNA was extracted from ear tissue of F0 generation individuals, and the target editing region was amplified by PCR. The amplification system was as follows: 100 ng template DNA, 0.4 μM each of forward and reverse primers, 200 μmol / L dNTPs, and 0.5 U high-fidelity Taq enzyme. The amplified products were initially screened by T7E1 enzyme digestion or high-resolution melting curve HRM. Positive samples were further subjected to TA cloning + Sanger sequencing or high-throughput sequencing amplicon-seq with a sequencing depth ≥1000×. Editing efficiency is defined as: the number of clones containing the edited allele in the test sample / the total number of clones × 100%, requiring an editing efficiency of ≥60% for a single F0 individual; Chimeras are classified as follows: if ≥2 different indel types are detected and the dominant type accounts for <80%, they are defined as highly chimeric and are not allowed to enter the breeding system; only low chimerism or quasi-homozygous F0 individuals with editing efficiency ≥60% and dominant edit type accounting for ≥80% are retained for subsequent breeding. Off-target detection uses whole-genome sequencing (WGS) with a sequencing depth ≥30 or targeted deep sequencing targeting the top 10 predicted off-target sites with a depth ≥1000. If the mutation frequency of any off-target site exceeds 0.5%, it is considered high-risk and the individual is excluded.

[0028] Step 3: Establishing a multi-generational pedigree Qualified F0 generation rams are mated with wild-type ewes of the same breed with the same genetic background and an inbreeding coefficient of <5% through natural mating or artificial insemination to obtain the F1 generation. Individuals carrying the target edited allele in the F1 generation were selected, confirmed by PCR and sequencing, and then crossed with siblings or half-sibs to obtain the F2 generation. In the F2 generation, homozygous edited individuals are selected and interbred to obtain the F3 generation; The number of individuals effectively involved in breeding in each generation shall not be less than 8, the sex ratio shall be close to 1:1, the inbreeding increment shall be controlled at <6.25% per generation to avoid full-sib mating, and the pedigree record shall include individual number, parent ID, date of birth, mating combination, genotype status, and record completeness rate ≥95%.

[0029] The target trait is muscle growth. Phenotypic assessment is conducted at four stages: birth, 90 days after weaning, 6 months of age, and 12 months of age. Measurement indicators include weight, daily weight gain, height, length, and chest circumference. Among them, the increase in daily weight gain is considered to be effective if it is not less than 15% in F1 heterozygotes and not less than 25% in F2 homozygotes.

[0030] Genotyping was performed using PCR amplification combined with high-resolution melting curve analysis (HRM) or next-generation sequencing (NGS). The sequencing depth of the target editing site was no less than 500×, and the genotyping accuracy was ≥99%. The genetic transmission rate of the target allele in three consecutive generations of F1–F3 was no less than 85% of the theoretical value, and there were no reversion mutations or new insertions / deletions (indels).

[0031] The phenotypic stability score P is calculated using the following formula: P=1-CV n / CV0 Among them, CV nCV0 is the phenotypic coefficient of variation for the target trait in the nth generation (n≥F1), and CV0 is the coefficient of variation for the wild-type control population. The P-value is normalized to the [0,1] interval. When the P-values ​​for F1–F3 generations are all ≥0.75, the phenotype is considered stable.

[0032] Genotype homology score G is determined as follows: G=N e / N t Where N e N represents the number of individuals in a generation that possess the expected target genotype, such as homozygous edited genotype. t The total number of individuals tested in this generation; a G value of ≥0.45 in the F2 generation meets the 1:2:1 segregation ratio, and a G value of ≥0.70 in the F3 generation is considered to indicate that the genotype is stabilizing.

[0033] The genetic concordance score H is calculated using a chi-square test, with the following formula: H=1-(X 2 / X 2 0.05 ) Where X 2 X is the chi-square value of the actual genotype segregation ratio versus the theoretical Mendelian segregation ratio. 2 0.05 The critical value is 5.99 when the degrees of freedom are 2; when H≥0.80, it is considered that the genetic law conforms to expectations.

[0034] The genetic stability index (SSI) is calculated using a weighted formula: SSI = 0.4 × P + 0.4 × G + 0.2 × H The weighting coefficients were determined by principal component analysis (PCA). When the average SSI of three consecutive generations F1–F3 is ≥0.80 and the lowest SSI of a single generation is ≥0.70, the target trait is considered to be genetically stable.

[0035] Off-target effect assessment was conducted in the F0, F2 and F3 generations. At least 20 potential off-target sites were predicted using GUIDE-seq or CIRCLE-seq, and their mutation frequencies were verified by deep sequencing. If the mutation frequency of any off-target site in any generation exceeded 1.0%, it was determined to be genetically unstable, and the propagation of the pedigree should be terminated.

[0036] Environmental factors control included: all individuals were housed in the same standardized sheepfold, with a temperature of 15–25℃, relative humidity of 50–70%, and fed a uniform complete feed, with daily feed intake variation not exceeding ±5%; phenotypic data collection was conducted by two or more technicians in a double-blind operation, with measurement errors controlled within ±2%.

[0037] Establish a genetic evaluation database based on SQL or MongoDB to store pedigree information, genotype data, phenotypic records and environmental parameters, gene frequency dynamic curves and SSI heatmaps; the evaluation period should be no less than 24 months, covering at least three reproductive cycles, and the data missing rate should be less than 5% for final stability determination.

[0038] The working principle of a method for assessing the genetic stability of target traits in gene-edited sheep is as follows: Precise introduction of gene-editing events and F0 generation screening. Using gene-editing tools such as CRISPR / Cas9, highly specific sgRNAs are designed for key functional genes in the sheep genome that regulate specific traits such as muscle growth, hair length, and disease resistance, including MSTN, FGF5, and PRNP. Targeted modification is achieved in fertilized eggs or somatic cells. F0 generation individuals are obtained through microinjection or somatic cell nuclear transfer techniques.

[0039] The Cas9-sgRNA complex recognizes and cleaves target DNA sites. Cells introduce insertions or deletions through the NHEJ repair mechanism of non-homologous end joining, leading to gene inactivation or alteration.

[0040] By using high-throughput sequencing to identify the genotype of the F0 generation, individuals with a high chimeric master edited genotype ratio of <80% and a high off-target mutation frequency of >0.5% were removed. This ensured that the F0 generation entering the breeding system had high editing efficiency, low chimerism, and genomic safety, providing a reliable starting point for subsequent genetic stability assessment.

[0041] The qualified F0 generation individuals are mated with wild-type sheep to obtain the F1 generation. Then, the F1 generation is obtained by interbreeding, and the F2 generation is obtained by interbreeding the homozygous F2 generation. This establishes a complete breeding pedigree of at least three generations.

[0042] If the target edited allele is a single-site mutation, the F1 generation should be a 1:1 segregation of heterozygotes, the F2 generation genotype ratio should be close to 1 homozygous edited: 2 heterozygous: 1 wild-type, and the F3 generation homozygous population tends to be fixed.

[0043] Tracking mechanism: Through pedigree records and genotyping, the transmission path of the target allele in each generation is dynamically tracked to identify whether gene silencing, reversion mutations, segregation bias, or genetic drift exist, thereby determining whether the editing site is stably inherited.

[0044] During key growth stages such as birth, weaning, 6 months of age, and adulthood, standardized phenotypic measurements of target traits, such as weight, daily weight gain, hair length, and disease resistance, were performed on each generation of individuals, and correlation analysis was conducted with genotype data.

[0045] If the target trait is dominated by the edited gene, individuals with the same genotype should exhibit similar phenotypes, and the edited individuals should exhibit significantly better phenotypes than wild-type individuals, such as MSTN knockout sheep with a ≥15% increase in daily weight gain.

[0046] By calculating the phenotypic coefficient of variation (CV), if the CV of the target trait continuously decreases and remains stable within 8% from F1 to F3, it indicates that the phenotypic expression tends to be consistent and has functional reproducibility and environmental adaptability.

[0047] The Stability Score Index (SSI) is introduced to integrate the three dimensions of phenotypic stability (P), genotypic consistency (G), and genetic concordance (H) with weights, thereby achieving standardized, comparable, and automated determination of the evaluation results.

[0048] Computational model: SSI = 0.4 × P + 0.4 × G + 0.2 × H in: P=1–CV n / CV0 represents phenotypic stability, where CV0 is the coefficient of variation in the wild-type population. G=N e / N t Genotypic consistency, such as the percentage of homozygous individuals in the F2 generation. H=1-(X 2 / X 2 0.05 ) Judgment logic: When the average SSI for three consecutive generations is ≥0.80 and there is no significant off-target inheritance or phenotypic degradation, the target trait is determined to be genetically stable and can enter the breeding application stage.

[0049] By identifying potential risks through data mining, such as a sudden increase in off-target frequency in a generation, or a decrease in penetrance due to deviation between phenotype and genotype, the system can automatically trigger a retest or terminate the propagation process.

[0050] The evaluation results guide the optimization of gene editing strategies, forming a closed-loop breeding system for editing evaluation and optimization.

[0051] Test case To verify the scientific validity, operability, and effectiveness of the present invention, "A Method for Evaluating the Genetic Stability of Target Traits in Gene-Edited Sheep," Hu sheep were selected as the experimental animal model. With muscle growth as the target trait, the MSTN gene and the inhibin-producing gene were edited using CRISPR / Cas9 technology to construct a gene-edited population. Genetic stability was then evaluated for three consecutive generations (F0–F3) according to the method described in this invention. The specific experimental process and results are as follows: I. Experimental Materials and Design laboratory animals Recipient sheep: Healthy adult Hu sheep with a clear genetic background and an inbreeding coefficient of <5%; 30 recipient ewes will be used for embryo transfer.

[0052] Editing target: Exon 1 region of the MSTN gene, GenBank: NC_019467.1, position g.1245–1265. Mutations in this region can lead to loss of gene function and promote muscle development.

[0053] sgRNA design and editing vectors sgRNA sequence: 5′-GCTGACCTTGGAGAAGGTGC-3′ Targets MSTN exon 1 The off-target score predicted by the CHOPCHOP tool was 2.1, and it was a unique match in the sheep genome.

[0054] Microinjection was performed using a complex of 50 ng / μL Cas9 mRNA and 25 ng / μL sgRNA.

[0055] Trial period: January 2022 – June 2024 (30 months) II. Experimental Procedure 1. Obtaining and screening F0 generation gene-edited sheep (January 2022 – June 2022) The Cas9 / sgRNA complex was microinjected into 120 single-cell stage Hu sheep zygotes and cultured in vitro to the blastocyst stage, yielding a total of 68 usable embryos.

[0056] The embryos were transplanted into 30 recipient ewes that were in estrus. The pregnancy rate was 60% (18 / 30), and 15 lambs were born in the F0 generation.

[0057] DNA was extracted from ear tissue, and the MSTN target region was amplified by PCR. Initial screening by T7E1 enzyme digestion showed that 9 animals were positive.

[0058] Further TA cloning + Sanger sequencing, with ≥20 clones detected per animal, showed the following results: Five individuals with editing efficiency ≥60% and primary editing type of 5 bp missing c.1_5del, accounting for ≥80%, were judged to be low chimerism; Whole-genome sequencing (WGS) at 30× depth detected the top 20 predicted off-target sites, with mutation frequencies all <0.5%. Ultimately, three F0 generation rams, numbered F0-03, F0-07, and F0-11, were selected to enter the breeding system.

[0059] 2. F1 Generation Population Construction and Preliminary Phenotypic Evaluation (July 2022 – January 2023) Three F0 generation rams were artificially inseminated with 12 wild-type Hu sheep ewes, resulting in a total of 21 F1 generation lambs.

[0060] Blood samples were collected at 90 days post-weaning for Sanger sequencing to identify genotypes. 10 are hybrid edit types + / − 11 are wild-type + / + The separation ratio was close to 1:1, χ²=0.024, P>0.05, which was in line with expectations.

[0061] At 6 months of age, growth traits such as weight, daily weight gain, and body length were measured. P<0.01, the difference is extremely significant. 3. Analysis of genetic segregation patterns in the F2 generation (February 2023 – August 2023) Six F1 generation heterozygotes (3 males and 3 females) were selected for sibling crosses to obtain 24 F2 generation lambs.

[0062] Genetic analysis results show: Purely edited type − / −: 6 Heterozygous + / −: 13 individuals Wild type + / +: 5 The actual ratio 6:13:5≈1:2.17:0.83, χ²=1.96<5.99df=2, α=0.05, which conforms to Mendel's law of segregation of 1:2:1.

[0063] Phenotypic determination at 6 months of age: A significant gene dosage effect was observed, with homozygous individuals showing a 29.3% increase in daily weight gain compared to wild-type individuals.

[0064] 4. F3 generation trait fixation and stability assessment 2023.09–2024.06 Six homozygous edited individuals from the F2 generation were crossbred to obtain 14 lambs from the F3 generation.

[0065] Genotyping: All 14 animals were homozygous edited type − / −, with no wild type or new indels, and genotypic concordance reached 100%.

[0066] Weight measurement at 12 months: Average weight: 78.6 ± 4.2 kg The coefficient of variation (CV) was 5.3% for the F1 generation and 11.8% for the F2 generation, indicating a high degree of phenotypic consistency.

[0067] Off-target monitoring: 15 predicted off-target sites were detected in F0, F2 and F3 generations. Deep sequencing ≥1000× showed that the mutation frequency of all sites was <0.4%, and there were no hereditary off-targets.

[0068] III. Calculation of the Genetic Stability Index (SSI) According to the formula described in claim 7 of this invention: SSI = 0.4 × P + 0.4 × G + 0.2 × H P=1–CV n / CV0CV0=11.5%; G=N e / N t H = 1 – χ² / 5.99 The F3 generation SSI=0.90≥0.80, and the phenotype is stable, there is no off-target inheritance, and the genotype is completely fixed, which meets the genetic stability determination criteria of this invention.

[0069] IV. Experimental Conclusions The method of this invention has successfully achieved three generations of genetic tracking of the MSTN gene-edited Hu sheep population; The target trait of muscle growth was consistently expressed in the F1–F3 generations and showed a dose-response relationship, with phenotypic variation gradually decreasing. The transmission of the target allele follows Mendelian inheritance laws, and the genotype is completely fixed in the F3 generation. Off-target effects were not inherited in offspring, and the genome safety was good; The average SSI for three consecutive generations was 0.76, and the SSI for the F3 generation was 0.90 (≥0.80), indicating that the muscle growth trait regulated by this gene editing event has excellent genetic stability.

[0070] V. Significance of the Experiment This experimental example verifies the feasibility and reliability of the method of the present invention in practical applications, providing a complete technical path and data support for the breeding, biosafety assessment, and industrialization of gene-edited sheep. This method can be extended to other economic traits such as wool quality, disease resistance, and the assessment of genetic stability in livestock species such as goats and cattle.

[0071] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A method for assessing the genetic stability of a target trait in gene-edited sheep, characterized in that, The specific steps include the following: Step 1: Construction of gene editing vector and acquisition of F0 generation individuals Gene editing vectors were constructed to specifically target functional genes in the sheep genome associated with the target trait using sgRNA. The functional genes are MSTN myostatin gene (GenBank accession number NC_019467.1), exon 1 region, PRNP prion protein gene, and genes related to anti-pruritus or FGF5 hair follicle development regulation. The sgRNA sequence is 20 bp in length and has a 5'-N... 18-22 The GG-3' structure was confirmed by BLAST alignment to have unique targeting in the sheep genome, with an off-target prediction score of ≤3 using COSMID or CHOPCHOP tools. The sgRNA and Cas9 mRNA or Cas9 protein complex were introduced into the pronucleus of single-cell fertilized eggs of New Zealand white sheep or Hu sheep via microinjection. The injection concentrations were: Cas9 mRNA 50 ng / μL, sgRNA 25 ng / μL, and the injection volume was 10 pL / egg. Alternatively, the somatic cell nuclear transfer (SCNT) method can be used: CRISPR / Cas9-edited sheep ear fibroblasts are used as donor cells and transplanted into enucleated MII stage oocytes. The fusion voltage is 1.2 kV / cm, the pulse duration is 30 μs, and activation is performed by ionomycin 5 μmol / L for 5 min combined with 6-DMAP 2 mmol / L for 4 h. After the embryos are cultured in vitro to the morula or blastocyst stage, they are transferred into the uterus of a recipient ewe in estrus. During pregnancy, ultrasound monitoring confirms that the embryos are developing normally, and F0 generation lambs are born. Step 2: Identification and Screening Criteria for F0 Generation Molecular Basis Genomic DNA was extracted from ear tissue of F0 generation individuals, and the target editing region was amplified by PCR. The amplification system was as follows: 100 ng template DNA, 0.4 μM each of forward and reverse primers, 200 μmol / L dNTPs, and 0.5 U high-fidelity Taq enzyme. The amplified products were initially screened by T7E1 enzyme digestion or high-resolution melting curve HRM. Positive samples were further subjected to TA cloning + Sanger sequencing or high-throughput sequencing amplicon-seq with a sequencing depth ≥1000×. Editing efficiency is defined as: the number of clones containing the edited allele in the test sample / the total number of clones × 100%, requiring an editing efficiency of ≥60% for a single F0 individual; Chimeras are classified as follows: if ≥2 different indel types are detected and the dominant type accounts for <80%, they are defined as highly chimeric and are not allowed to enter the breeding system; only low chimerism or quasi-homozygous F0 individuals with editing efficiency ≥60% and dominant edit type accounting for ≥80% are retained for subsequent breeding. Off-target detection uses whole-genome sequencing (WGS) with a sequencing depth ≥30 or targeted deep sequencing targeting the top 10 predicted off-target sites with a depth ≥1000. If the mutation frequency of any off-target site exceeds 0.5%, it is considered high-risk and the individual is excluded. Step 3: Establishing a multi-generational pedigree Qualified F0 generation rams are mated with wild-type ewes of the same breed with the same genetic background and an inbreeding coefficient of <5% through natural mating or artificial insemination to obtain the F1 generation. Individuals carrying the target edited allele in the F1 generation were selected, confirmed by PCR and sequencing, and then crossed with siblings or half-sibs to obtain the F2 generation. In the F2 generation, homozygous edited individuals are selected and interbred to obtain the F3 generation; The number of individuals effectively involved in breeding in each generation shall not be less than 8, the sex ratio shall be close to 1:1, the inbreeding increment shall be controlled at <6.25% per generation to avoid full-sib mating, and the pedigree record shall include individual number, parent ID, date of birth, mating combination, genotype status, and record completeness rate ≥95%.

2. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, The target trait is muscle growth. Phenotypic assessment is conducted at four stages: birth, 90 days after weaning, 6 months of age, and 12 months of age. Measurement indicators include weight, daily weight gain, height, length, and chest circumference. Among them, the increase in daily weight gain is considered to be effective if it is not less than 15% in F1 heterozygotes and not less than 25% in F2 homozygotes.

3. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, Genotyping was performed using PCR amplification combined with high-resolution melting curve analysis (HRM) or next-generation sequencing (NGS). The sequencing depth of the target editing site was no less than 500×, and the genotyping accuracy was ≥99%. The genetic transmission rate of the target allele in three consecutive generations of F1–F3 was no less than 85% of the theoretical value, and there were no reversion mutations or new insertions / deletions (indels).

4. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, The phenotypic stability score P is calculated using the following formula: P=1-CV n / CV0 Among them, CV n CV0 is the phenotypic coefficient of variation for the target trait in the nth generation (n≥F1), and CV0 is the coefficient of variation for the wild-type control population. The P-value is normalized to the [0,1] interval. When the P-values ​​for F1–F3 generations are all ≥0.75, the phenotype is considered stable.

5. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, Genotype homology score G is determined as follows: G=N e / N t Where N e N represents the number of individuals in a given generation that possess the desired target genotype. t The total number of individuals tested is considered; a G value of ≥0.45 in F2 generation indicates a 1:2:1 segregation ratio, and a G value of ≥0.70 in F3 generation indicates that the genotype is stabilizing.

6. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, The genetic concordance score H is calculated using a chi-square test, with the following formula: H=1-(X 2 / X 2 0.05 ) Where X 2 X is the chi-square value of the actual genotype segregation ratio versus the theoretical Mendelian segregation ratio. 2 0.05 The critical value is 5.99 when the degrees of freedom are 2; when H≥0.80, it is considered that the genetic law conforms to expectations.

7. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, The genetic stability index (SSI) is calculated using a weighted formula: SSI = 0.4 × P + 0.4 × G + 0.2 × H The weighting coefficients were determined by principal component analysis (PCA). When the average SSI of three consecutive generations F1–F3 is ≥0.80 and the lowest SSI of a single generation is ≥0.70, the target trait is considered to be genetically stable.

8. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, Off-target effect assessment was conducted in the F0, F2 and F3 generations. At least 20 potential off-target sites were predicted using GUIDE-seq or CIRCLE-seq, and their mutation frequencies were verified by deep sequencing. If the mutation frequency of any off-target site in any generation exceeded 1.0%, it was determined to be genetically unstable, and the propagation of the pedigree should be terminated.

9. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, Environmental factor control includes: all individuals are raised in the same standardized sheepfold, with a temperature of 15–25℃, relative humidity of 50–70%, fed with uniform complete feed, and the daily average feed intake varies by no more than ±5%; measurement error is controlled within ±2%.

10. The method for assessing the genetic stability of a target trait in gene-edited sheep according to claim 1, characterized in that, Establish a genetic evaluation database based on SQL or MongoDB to store pedigree information, genotype data, phenotypic records and environmental parameters, gene frequency dynamic curves and SSI heatmaps; the evaluation period should be no less than 24 months, covering at least three reproductive cycles, and the data missing rate should be less than 5% for final stability determination.

Images