Mouse Oct4 conditional gene knockout model construction method based on quadruple sgRNA collaborative targeting

Abstract

The invention relates to the technical field of construction of conditional gene knockout models, and discloses a construction method of a mouse Oct4 conditional gene knockout model based on quadruple sgRNA collaborative targeting, which comprises the following steps: step 1, designing four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4, in which the sgRNA1 and the sgRNA2 target 5'end and 3 'end non-coding regions of a second exon of an Oct4 gene, and the sgRNA3 and the sgRNA4 target structural domain conserved sequences; step 2, preparing an electrotransfection solution, wherein the electrotransfection solution is composed of Cas9mRNA, the four sgRNAs and an electrotransfection buffer solution; step 3, carrying out electroporation treatment on the fertilized eggs of the mice; step 4, culturing the fertilized eggs subjected to the electrotransformation treatment to a blastocyst stage; and 5, carrying out genotype identification on the cultured blastocyst. According to the scheme, the sgRNA combination and embryo electrotransfection system is optimized, the mouse Oct4 conditional gene knockout model is efficiently and accurately constructed, a reliable tool is provided for regulation mechanism research of Oct4 in mouse early embryo trigerm differentiation, and the medical transformation value of Oct4 is excavated.

Description

Technical Field

[0001] This invention relates to the field of conditional gene knockout model construction technology, specifically to a method for constructing a mouse Oct4 conditional gene knockout model based on quadruple sgRNA synergistic targeting. Background Technology

[0002] The construction of a conditional Oct4 knockout model in mice is a core tool for elucidating the function of the Oct4 gene, exploring embryonic development mechanisms, and advancing regenerative medicine research. Oct4, a key transcription factor in the POU family, is a core molecule for maintaining embryonic stem cell pluripotency and regulating early embryonic development. It is widely involved in the regulation of blastocyst inner cell mass characteristics and somatic cell reprogramming. Abnormal Oct4 function is closely related to abnormal embryonic development and disordered stem cell differentiation. Existing Oct4 full knockout models have extremely high embryo lethality rates, making it difficult to obtain surviving individuals. Conditional knockout models, however, can achieve spatiotemporally specific knockout of the Oct4 gene at specific germ layers and developmental stages, effectively avoiding embryonic lethality and precisely elucidating its dynamic regulatory function. This plays an irreplaceable role in the study of Oct4-related molecular mechanisms and the translational application of regenerative medicine, while also providing reliable animal models to support the study of the pathogenesis and targeted therapy of related diseases.

[0003] In existing technologies, to improve the efficiency and accuracy of constructing mouse Oct4 conditional gene knockout models, various technical approaches have emerged, focusing on gene editing technology optimization, process improvement, and risk control: First, optimizing traditional homologous recombination technology by improving vector construction strategies and optimizing screening methods to increase the positive clone rate and shorten the model construction cycle; second, applying CRISPR / Cas9 gene editing technology to replace traditional homologous recombination technology to simplify the operation process, and improving targeted editing efficiency by optimizing sgRNA design and adjusting Cas9 protein delivery methods; third, employing single or double sgRNA-mediated editing strategies to improve target specificity and reduce off-target risks; fourth, improving the model screening and validation system by using steps such as genotype identification and off-target detection to enhance model construction accuracy and reduce the impact of non-specific mutations on model accuracy; and fifth, optimizing embryo injection and transfer processes to improve the embryo survival environment, reduce embryo lethality, and increase the rate of obtaining surviving model individuals.

[0004] However, existing technologies for improving the efficiency and accuracy of constructing mouse Oct4 conditional gene knockout models still suffer from the following technical problems, making it difficult to meet the needs of scientific research and translational applications: First, the construction efficiency is low and the cycle is lengthy. Traditional homologous recombination technology has a construction cycle of 12-18 months, with a positive clone rate of less than 10%. Even with conventional CRISPR / Cas9 technology, the limitations of single sgRNA design lead to large fluctuations in editing efficiency (25-75%), making it impossible to achieve stable and efficient large-scale model construction. Second, the editing accuracy is insufficient and the off-target risk is high. When the existing CRISPR / Cas9 system is applied to Oct4 gene editing, the average off-target rate reaches 5-20%, and the off-target effects can lead to... Non-specific gene mutations can compromise model accuracy and interfere with the precise analysis of Oct4 gene function. Thirdly, weak embryo viability and difficulty in achieving specific knockout pose challenges; existing Oct4 full knockout models have an embryo lethality rate exceeding 80%, and conditional knockout models lack effective spatiotemporal specific regulatory mechanisms, making it impossible to precisely achieve gene knockout at specific germ layers or developmental stages, thus hindering the analysis of Oct4's dynamic regulatory mechanisms. Fourthly, the model validation and application system is incomplete, lacking a systematic and rigorous process for genotyping, off-target analysis, and phenotypic detection, making it impossible to fully verify the accuracy and stability of model editing. Furthermore, the models struggle to dynamically track Oct4 gene function, limiting their application value in mechanistic research and translational medicine. In summary, current technologies cannot simultaneously achieve high efficiency, accuracy, and practicality in model construction, necessitating a novel and efficient construction method to overcome existing technological bottlenecks and provide reliable support for Oct4 gene function research. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a mouse Oct4 conditional gene knockout model based on quadruple sgRNA synergistic targeting, in order to solve the technical problems of long model construction cycle, low editing efficiency, high off-target rate and high embryo lethality when constructing mouse Oct4 conditional gene knockout models in the existing technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for constructing a mouse Oct4 conditional gene knockout model based on quadruple sgRNA synergistic targeting, comprising the following steps: Step 1: Design four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4. Among them, sgRNA1 and sgRNA2 target the 5' end and 3' end uncoding regions of the second exon of the Oct4 gene, and sgRNA3 and sgRNA4 target conserved domain sequences. Step 2: Prepare the electroporation buffer, which consists of Cas9 mRNA, the aforementioned four sgRNAs, and electroporation buffer. Step 3: Electroporation of mouse fertilized eggs; Step 4: Culture the electroporated fertilized eggs to the blastocyst stage; Step 5: Genotyping the blastocysts obtained from the culture.

[0007] Preferably, as an improvement, in step one, the GC content of the four sgRNAs is 40-60% to avoid the occurrence of a sequence structure with four consecutive Ts; the off-target score of the four sgRNAs is ensured to be >90 by using the MIT_CRISPR design tool.

[0008] Preferably, as an improvement, in step one, the nucleotide sequence of the Oct4 gene is as shown in SEQ_ID_NO.1, and the nucleotide sequences of the four sgRNAs are as follows: sgRNA1 is as shown in SEQ_ID_NO.2, sgRNA2 is as shown in SEQ_ID_NO.3, sgRNA3 is as shown in SEQ_ID_NO.4, and sgRNA4 is as shown in SEQ_ID_NO.5.

[0009] Preferably, as an improvement, in step two, the electroporation buffer comprises the following components and final concentrations: Cas9 mRNA 50 ng / μl, 4 sgRNAs 25 nM each, and 20% electroporation buffer; the electroporation buffer comprises the following components and final concentrations: HEPES 100 mM, KCl 750 mM, MgCl2·6H2O 5 mM, TCEP 5 mM, Glycerol 50%, pH adjusted to 7.5 with KOH, and the remainder being ddH2O.

[0010] Preferably, as an improvement, in step three, the conditions for the electro-transfer processing are: voltage 30V, pulses 4-6 times, pulse length 3ms, and pulse interval 100ms.

[0011] Preferably, as an improvement, in step four, the culture is carried out in a constant temperature incubator at 37°C and 5% CO2 until the embryo develops to the blastocyst stage.

[0012] Preferably, as an improvement, in step five, the genotype identification includes (1) primary screening by amplifying the second exon editing region of the Oct4 gene using nested PCR; (2) in-depth verification by ChIP-Seq sequencing combined with 0.025 restriction endonuclease detection; and (3) off-target analysis by detecting predicted off-target sites through whole-genome sequencing.

[0013] Preferably, as an improvement, the primers used in the primary screening include outer primer F1 / R1 and inner primer F2 / R2, wherein the nucleotide sequence of outer primer F1 is shown in SEQ_ID_NO.6, the nucleotide sequence of outer primer R1 is shown in SEQ_ID_NO.7, the nucleotide sequence of inner primer F2 is shown in SEQ_ID_NO.8, and the nucleotide sequence of inner primer R2 is shown in SEQ_ID_NO.9.

[0014] Preferably, as an improvement, this scheme also provides a mouse Oct4 conditional gene knockout model, which is constructed by the above method. The fertilized egg development rate of the constructed Oct4 conditional gene knockout mouse model is ≥85%, the gene editing efficiency is ≥85%, and the off-target rate is ≤1.5%.

[0015] Preferably, as an improvement, this scheme also provides an application of a mouse Oct4 conditional gene knockout model in studying the dynamic regulatory mechanism of differentiation of the three germ layers in early mouse embryos and assessing the embryo lethal risk associated with mutations in the human homolog Oct4L.

[0016] The principles and advantages of this scheme are: 1. Target Specificity: A combination of four synergistic sgRNAs was designed to precisely target key regions of the Oct4 gene. The design parameters were strictly controlled and validated for specificity, reducing the risk of off-target effects from the source.

[0017] 2. High operational efficiency: Optimized embryo electroporation solution formulation and electroporation parameters, combined with exclusive culture conditions, significantly improve the success rate of fertilized eggs developing into blastocysts and the efficiency of gene editing.

[0018] 3. Validation System: Establish a complete system of "primary screening - in-depth validation - off-target analysis - phenotypic tracking" to ensure the accuracy, stability and reliability of the model. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the Oct4 gene knockout strategy in an embodiment of the present invention. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0021] Overview of the Plan This method provides a technique for constructing a mouse Oct4 conditional gene knockout model based on the synergistic targeting of four sgRNAs. The core of this approach is to optimize the sgRNA combination design, embryo electroporation system, and validation analysis system to achieve efficient and accurate construction of the mouse Oct4 conditional gene knockout model. The specific steps are as follows: Step 1: Target sequence screening: Screening for 4 sgRNAs that synergistically target the Oct4 gene, such as Figure 1 As shown, sgRNA1 (i.e., sg1) and sgRNA2 (i.e., sg2) target the 5' and 3' uncoding regions of the second exon, respectively, and are within ±50 bp of the splice site; sgRNA3 (i.e., sg3) and sgRNA4 (i.e., sg4) target the conserved sequence of the Oct4 gene, which has been verified by the PhyloCSF algorithm to ensure targeting specificity.

[0022] The complete nucleotide sequence of the Oct4 gene is as follows (SEQ_ID_NO.1): .

[0023] Specifically, the GC content of sgRNA needs to be strictly controlled between 40% and 60% to avoid the occurrence of a sequence structure with 4 consecutive Ts; the off-target score of all 4 sgRNAs is ensured to be >90 by using the MIT_CRISPR design tool to reduce the risk of off-target effects.

[0024] Four sgRNAs were synthesized according to the above design parameters, purified, and used separately. The specific sgRNA sequences are as follows: sg1 (5'-CCCATGAATTATTACTGACC-3', as shown in SEQ_ID_NO.2), sg2 (5'-GGGTCACGGTCAAATGGTAT-3', as shown in SEQ_ID_NO.3), sg3 (5'-CAAAGGCTCTACATCTCGGA-3', as shown in SEQ_ID_NO.4), and sg4 (5'-CCTTGAGGTGGCTCAACCAT-3', as shown in SEQ_ID_NO.5).

[0025] Step 2: Electroporation buffer preparation: Prepare the electroporation buffer according to the optimized formula. The final concentration is: Cas9 mRNA 50 ng / μl + each of the above 4 sgRNAs 25 nM + 20% electroporation buffer. The electroporation buffer includes the following components and final concentrations: HEPES 100 mM, KCl 750 mM, MgCl2·6H2O 5 mM, TCEP 5 mM, Glycerol 50%, pH adjusted to 7.5 with KOH, and the remainder is ddH2O.

[0026] In addition, it also includes obtaining fertilized eggs and pretreatment: healthy adult mice are selected, and fertilized eggs are obtained after superovulation treatment. Fertilized eggs with intact morphology are screened, washed three times with serum-free culture medium to remove impurities, and then used for later use.

[0027] Step 3, embryo electroporation: Add the pretreated fertilized eggs to the prepared electroporation solution and set the optimized electroporation parameters: voltage 30V, pulse 4-6 times, pulse length 3ms, pulse interval 100ms to ensure embryo survival rate during electroporation; after electroporation, wash the fertilized eggs twice with KSOM medium (specific components and concentrations are shown in Table 1) to remove residual electroporation solution.

[0028] Step 4, Embryo Culture: Place the electroporated fertilized eggs in KSOM medium and culture them in a constant temperature incubator at 37℃ and 5% CO2 until they develop into blastocysts. Maintain a stable culture environment throughout the process and avoid contamination.

[0029] Table 1. Specific components and concentrations of KSOM culture medium

[0030] Step 5: Genotyping: (1) Primary screening: The second exon editing region of the Oct4 gene was amplified by PCR technology. The outer primers were F1 / R1 (amplification product 8000 bp) and the inner primers were F2 / R2 (amplification product 500-1100 bp). Gene editing positive embryos were preliminarily screened by agarose gel electrophoresis.

[0031] The nucleotide sequence of the outer primer F1 is shown in SEQ_ID_NO.6 (5'-GTCTAGGACCTGACAAGTTGC-3'), the nucleotide sequence of the outer primer R1 is shown in SEQ_ID_NO.7 (5'-CTGACTGAAGCATGCAGTCAG-3'), the nucleotide sequence of the inner primer F2 is shown in SEQ_ID_NO.8 (5'-ACCTGGCACTCTGGCCTAG-3'), and the nucleotide sequence of the inner primer R2 is shown in SEQ_ID_NO.9 (5'-GGGGACAGAGGGGAAAACAA-3').

[0032] (2) In-depth verification: For embryos that are positive in the primary screening, ChIP-Seq sequencing combined with 0.025% restriction endonuclease detection is used to verify the accuracy and targeting of gene editing and confirm that the editing site meets the expectations.

[0033] (3) It also includes off-target analysis: extract genomic DNA from positive embryos, perform whole-genome sequencing, focus on detecting the Top 10 predicted off-target sites, confirm that the off-target rate is controlled at ≤1.5%, and ensure the accuracy of the model.

[0034] If editing efficiency, embryo development, or off-target rate does not meet expectations during this stage, it can be addressed through: (1) Adjusting the concentration and ratio of sgRNA, optimizing electroporation parameters, changing the electroporation solution formula, reducing the concentration of Cas9 mRNA, and improving the embryo culture environment are all ways to improve embryo survival rate and editing efficiency. (2) By using nested PCR for preliminary screening, TA cloning and sequencing, ChIP-Seq verification and whole genome sequencing, it is possible to accurately confirm whether the editing site is consistent with the expectation and detect off-target situations. Specifically, this scheme uses 30× coverage whole genome resequencing to perform whole genome-wide variation scanning on the edited embryo samples. At the same time, PCR amplification and Sanger sequencing verification are performed on the Top 10 high-risk potential off-target sites predicted by the MITCRISPR tool. Combined with bioinformatics analysis and statistical calculations, the off-target rate of this technology system is finally determined to be ≤1.5%.

[0035] (3) Subsequent experiments will only select positive embryos with accurate editing sites, no obvious off-target effects, and clear genotypes for further culture and establishment to ensure the reliability of the model and the accuracy of subsequent research data.

[0036] Finally, it also includes phenotypic analysis and model validation of the mouse Oct4 conditional gene knockout model. 1. Embryo development tracking: Using a time-resolved microscopic imaging system, embryo morphological changes are recorded every 6 hours to track the development process of the embryo from fertilized egg to blastocyst, and the success rate of embryo development to blastocyst is calculated to ensure ≥85%.

[0037] 2. Detection of marker genes in the three germ layers: The expression levels of the ectoderm marker gene SOX2, the mesoderm marker gene Brachyury, and the endoderm marker gene FOXA2 in the embryo were quantitatively detected by qRT-PCR technology to analyze the effect of Oct4 knockout on the differentiation of the three germ layers.

[0038] 3. Protein localization analysis: The Oct4 antibody immunofluorescence staining method was used to observe the subcellular distribution of Oct4 protein through confocal microscopy, clarifying the changes in protein expression and localization after Oct4 gene knockout, and completing the comprehensive validation of the model.

[0039] Example 1 120 mouse fertilized eggs were selected and edited using the aforementioned quadruple sgRNA combination (sg1-sg4) and an optimized electroporation system (30V, 4-6 pulses). The embryos were cultured in KSOM medium to the blastocyst stage. The core design and electroporation parameters employed in this patented method were used, supplemented by PCR and whole-genome sequencing for verification. The results were significant: the targeted editing success rate reached 85.3%, the off-target rate was only 1.2%, and the blastocyst development rate was 82%. This completely solves the problem of over 80% embryo lethality in traditional full knockout models, successfully obtaining a conditional knockout model suitable for tri-germ layer differentiation research.

[0040] In summary, this invention significantly shortens the model construction cycle, improves operational efficiency, and reduces time and material costs. It achieves high gene editing efficiency and extremely low off-target rates, resulting in a substantial increase in embryo survival rates and effectively solving the problems of high lethality and poor specificity associated with traditional techniques. The model is stable and reliable, enabling precise conditional knockout, providing a safer, more efficient, and practical tool for studying embryonic development mechanisms. Overall, the technical performance is significantly superior to existing methods.

[0041] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs, characterized in that: Includes the following steps: Step 1: Design four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4. Among them, sgRNA1 and sgRNA2 target the 5' end and 3' end uncoding regions of the second exon of the Oct4 gene, and sgRNA3 and sgRNA4 target conserved domain sequences. Step 2: Prepare the electroporation buffer, which consists of Cas9 mRNA, the aforementioned four sgRNAs, and electroporation buffer. Step 3: Electroporation of mouse fertilized eggs; Step 4: Culture the electroporated fertilized eggs to the blastocyst stage; Step 5: Genotyping the blastocysts obtained from the culture.

2. The method for constructing a mouse Oct4 conditional gene knockout model based on quadruple sgRNA synergistic targeting according to claim 1, characterized in that: In step one, the GC content of the four sgRNAs is 40-60% to avoid the occurrence of a sequence structure with four consecutive Ts; the off-target score of the four sgRNAs is ensured to be >90 by using the MIT_CRISPR design tool.

3. The method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 2, characterized in that: In step one, the nucleotide sequence of the Oct4 gene is shown in SEQ_ID_NO.1, and the nucleotide sequences of the four sgRNAs are as follows: sgRNA1 is shown in SEQ_ID_NO.2, sgRNA2 is shown in SEQ_ID_NO.3, sgRNA3 is shown in SEQ_ID_NO.4, and sgRNA4 is shown in SEQ_ID_NO.

5.

4. The method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 1, characterized in that: In step two, the electroporation buffer comprises the following components and final concentrations: Cas9 mRNA 50 ng / μl, 4 sgRNAs 25 nM each, and 20% electroporation buffer; the electroporation buffer comprises the following components and final concentrations: HEPES 100 mM, KCl 750 mM, MgCl2·6H2O 5 mM, TCEP 5 mM, Glycerol 50%, pH adjusted to 7.5 with KOH, and the remainder being ddH2O.

5. The method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 1, characterized in that: In step three, the conditions for the electro-spinning process are: voltage 30V, 4-6 pulses, pulse length 3ms, and pulse interval 100ms.

6. The method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 1, characterized in that: In step four, the culture is carried out in a constant temperature incubator at 37°C and 5% CO2 until the embryo develops to the blastocyst stage.

7. The method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 1, characterized in that: In step five, the genotype identification includes (1) primary screening by amplifying the second exon editing region of the Oct4 gene using nested PCR; (2) in-depth verification by ChIP-Seq sequencing combined with 0.025 restriction endonuclease detection; and (3) off-target analysis by detecting predicted off-target sites through whole-genome sequencing.

8. A method for constructing a mouse Oct4 conditional gene knockout model based on synergistic targeting of four sgRNAs according to claim 7, characterized in that: The primers used in the primary screening include outer primer F1 / R1 and inner primer F2 / R2. The nucleotide sequence of outer primer F1 is shown in SEQ_ID_NO.6, the nucleotide sequence of outer primer R1 is shown in SEQ_ID_NO.7, the nucleotide sequence of inner primer F2 is shown in SEQ_ID_NO.8, and the nucleotide sequence of inner primer R2 is shown in SEQ_ID_NO.

9.

9. A mouse Oct4 conditional gene knockout model, characterized in that, The Oct4 conditional gene knockout mouse model constructed by the method described in any one of claims 1 to 8 has a blastocyst development rate of ≥85%, a gene editing efficiency of ≥85%, and an off-target rate of ≤1.5%.

10. The application of the mouse Oct4 conditional gene knockout model according to claim 9 in studying the dynamic regulatory mechanism of differentiation of the three germ layers in early mouse embryos and assessing the embryonic lethal risk associated with mutations in the human homolog Oct4L.

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