A pair of sgRNAs targeting mouse FAM50A gene and application thereof

Abstract

This invention provides a pair of targeted mice FAM50A The study of gene sgRNAs and their applications falls under the field of gene editing technology. This includes sgRNA1 and sgRNA2, whose recognition sites are located in mice. FAM50A The third and fourth exon regions of the gene, with the sgRNA1 sequence shown in SEQ ID NO.1 and the sgRNA2 sequence shown in SEQ ID NO.2. Based on the targeting provided by this invention... FAM50A The sgRNA1 and sgRNA2 genes, when used together via the CRISPR / Cas9 system, can be efficiently used to construct stable knockouts. FAM50A The NIH / 3T3 and β-TC-6 cell lines of the gene are for FAM50A FAM50A It provides research tools and materials for gene function research and the development of related disease models.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to a pair of targeted mice. FAM50A The sgRNA of a gene and its application in the construction of gene knockout cell lines. Background Technology

[0002] FAM50A (Family with sequence similarity 50, member A), also known as Xchromosome-associated protein 5 ( XAP FAM50A is a highly conserved X-chromosome-linked gene widely expressed in various mammalian tissues. Studies have shown that abnormal function of FAM50A is closely related to hereditary diseases such as intellectual disability, developmental delay, and male infertility, as well as the development and progression of malignant tumors such as liver cancer and colon cancer. Therefore, elucidating the function of FAM50A is of great significance for understanding its biological role and the pathogenesis of related diseases.

[0003] CRISPR / Cas9 gene editing technology is a tool that relies on sgRNA to guide the Cas9 nuclease to target specific DNA sequences. It can achieve gene knockout or modification by inducing double-strand breaks and utilizing the cell's own non-homologous end joining (NHEJ) or homologous recombination (HDR) mechanisms. Notably, Cas9 and sgRNA only need to be expressed and act on the cell for a short time, without long-term integration into the genome to complete the editing process. The effectiveness of CRISPR / Cas9 technology is highly dependent on the design and selection of sgRNA. Existing research shows that a single sgRNA design may not achieve complete knockout in some cases due to insufficient editing efficiency; therefore, using multiple sgRNAs in combination has become a common strategy to improve knockout efficiency. However, the combined use of multiple sgRNAs often leads to large gene fragment deletions or unexpected genomic rearrangements, posing potential safety risks in research and preclinical applications. Furthermore, while traditional integrative expression methods using viral vectors can effectively introduce foreign sequences into the CRISPR / Cas9 system, they easily result in long-term residues of foreign sequences in the cellular genome, thus affecting the genetic stability of cell models and the reliability of subsequent applications.

[0004] Currently, there are no known treatments for mouse-derived cell lines. FAM50A There is a lack of publicly available reports on efficient and specific sgRNA pairs for gene knockout and their accompanying recombinant expression vectors, as well as a lack of cell lines that completely silence the FAM50A protein through non-integrative methods. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a pair of targeted mice FAM50A The study aims to address the lack of reliable mouse-derived cell lines by studying the sgRNA of genes and its application in constructing gene knockout cell lines. FAM50A Technical issues with gene knockout cell models.

[0006] In a first aspect, the present invention provides a pair of targeted mice FAM50A The sgRNA of genes includes:

[0007] sgRNA1: 5'-GGCAAAAAAGGAGCAGTCAA-3' (SEQ ID NO.1, located at...) FAM50A (the third exon region of the gene).

[0008] sgRNA2: 5'-AGAGATGGCTATGTACGAGG-3' (SEQ ID NO.2, located at...) FAM50A (Region of the 4th exon of the gene).

[0009] Secondly, the present invention provides a recombinant expression vector containing the above-mentioned sgRNA, the construction method of which is as follows:

[0010] The sequence of sgRNA1 was cloned into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid to obtain the recombinant expression vector pU6-sgRNA1;

[0011] The sgRNA2 sequence was cloned into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid to obtain the recombinant expression vector pU6-sgRNA2.

[0012] In some embodiments of the present invention, site-directed mutagenesis is employed, and the sequences of sgRNA1 and sgRNA2 are integrated into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid using primers shown in SEQ ID NO.3-4 and SEQ ID NO.5-6, respectively, thereby obtaining the recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2.

[0013] Thirdly, this invention provides the above-mentioned sgRNA and its recombinant expression vector for constructing FAM50A The application of gene knockout cell lines specifically includes the following steps:

[0014] S1. The sgRNA1 and sgRNA2 sequences were cloned into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid, respectively, to obtain the recombinant expression vector pU6-sgRNA1 containing the sgRNA1 sequence and the recombinant expression vector pU6-sgRNA2 containing the sgRNA2 sequence.

[0015] S2. Recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2 were electrotransfected into mouse cells, and positive monoclonal antibodies were screened.

[0016] S3. Identification revealed complete loss of FAM50A protein expression. FAM50A Gene knockout mouse cell lines.

[0017] In the above applications, mouse cells include the NIH / 3T3 cell line and the β-TC-6 cell line.

[0018] Preferably, in the above applications, the primers shown in SEQ ID NO.7-10 are used for identification.

[0019] Fourthly, the method constructed according to the present invention FAM50A Gene knockout cell lines also fall within the scope of this invention, and can be used for... FAM50A In fields such as gene function research, tumor mechanism research, and drug development.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention provides a pair of specific targets FAM50A The gene's sgRNA, combined with the CRISPR / Cas9 system, can achieve efficient and complete delivery in mouse-derived cells. FAM50A Gene knockout. The sgRNAs designed in this invention induce small insertions or deletions only near the target site, without disrupting the overall genome structure. This avoids the large-segment genome deletions common in traditional multi-sgRNA strategies, offering advantages such as precise targeting and high knockout efficiency. Furthermore, this invention utilizes electroporation technology to achieve transient expression of the CRISPR / Cas9 system in host cells. This method constructs a cell model with complete FAM50A protein deletion without requiring the selection of cell lines that stably integrate exogenous plasmids. It effectively avoids exogenous sequence residues caused by viral vector integration, thereby ensuring the genetic stability, safety, and reliability of the constructed gene-edited cell lines in subsequent research applications.

[0022] This invention successfully knocked out [the virus] in the NIH / 3T3 cell line and the β-TC-6 cell line. FAM50A The gene was knocked out, and experimental results demonstrated that this knockout method is safe, reliable, and does not cause large-scale deletions of genomic segments. This invention provides... FAM50A Gene knockout methods are highly practical and provide... FAM50A The study of gene function and the pathogenesis of related diseases, as well as the development of potential treatment strategies, provide effective methods and a foundation. Attached Figure Description

[0023] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0024] Picture 1 This is a map of the recombinant expression vector pU6-sgRNA1 in Example 1 of the present invention;

[0025] Picture 2 This is a map of the recombinant expression vector pU6-sgRNA2 in Example 1 of the present invention;

[0026] Picture 3 This is a sequencing peak diagram of the recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2 in Example 1 of the present invention;

[0027] Picture 4 The results of genotyping of NIH / 3T3 and β-TC-6 positive monoclonal cells in Example 2 of this invention;

[0028] Picture 5 The results of Western blot detection of FAM50A protein in NIH / 3T3 and β-TC-6 positive monoclonal cells in Example 2 of this invention are shown. Detailed Implementation

[0029] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion.

[0031] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0032] Example 1

[0033] This example provides a pair of CRISPR / Cas9 gene knockout vectors, and the construction process is as follows:

[0034] (1) Targeted mice FAM50A The sgRNA of a gene.

[0035] Based on the NCBI database FAM50A Gene sequence information (Gene ID: 108160), this example provides two targeted mice. FAM50A The gene's sgRNA, of which sgRNA1 targets FAM50A The third exon region of the gene, targeted by sgRNA2 FAM50A The specific sequence of the fourth exon region of the gene is shown below:

[0036] sgRNA1: 5'-GGCAAAAAAGGAGCAGTCAA-3' (SEQ ID NO.1);

[0037] sgRNA2: 5'-AGAGATGGCTATGTACGAGG-3' (SEQ ID NO.2).

[0038] (2) Construction of pU6-sgRNA recombinant expression vector.

[0039] The sgRNA1 and sgRNA2 sequences were integrated into the Cas9 / sgRNA co-expression vector pU6-sgROSA-1-CBh-Cas9-T2A-BFP (addgene: 64216) using site-directed mutagenesis to construct the recombinant expression vector. The procedure is as follows:

[0040] ① Design and synthesize primers for site-directed mutagenesis, as shown in Table 1.

[0041] Table 1. Primer sequences for point mutations corresponding to sgRNA1 and sgRNA2

[0042]

[0043] ② Preparation of linearized carriers.

[0044] The vector was amplified by reverse PCR using Phanta Max Super-Fidelity DNA Polymerase (Vazyme #P505), and the reaction system is shown in Table 2 and the reaction conditions are shown in Table 3.

[0045] Table 2 Reverse PCR reaction system

[0046]

[0047] Table 3 Reverse PCR reaction conditions

[0048]

[0049] ③ Amplification products Dpn I digest.

[0050] Add 1 μl to the amplification product from step ② Dpn I. The mixture was placed at 37°C for 2 h to digest and remove the methylated template plasmid from the product.

[0051] ④ In vitro recombination of linearized vectors.

[0052] The digested linearized carrier was cyclized and recombined in vitro using Exnase II catalytic enzyme. The reaction system is shown in Table 4.

[0053] Table 4. In vitro cyclization reaction system of linearized carrier

[0054]

[0055] After the reaction system was prepared, it was placed at 37°C for 30 min and then immediately placed on ice to cool.

[0056] ⑤ Transformation of recombinant expression plasmids.

[0057] Take DH5α competent cells stored at -80℃ and thaw them on ice. Then, take 100 μl of competent cells, add 10 μl of the recombinant reaction ligation product obtained in step ④, mix well, and incubate on ice for 30 min. Heat shock at 42℃ for 45 sec, then immediately cool on ice for 3 min. Add 900 μl of antibiotic-free LB medium and incubate on a shaker at 37℃ for 1 h (200-250 rpm). Spread on LB plates containing AMP (100 μg / mL) and incubate overnight at 37℃. Pick positive clones, incubate overnight on a shaker at 37℃, and then sequence. Samples with correctly sequenced bacterial cultures were preserved and plasmids extracted. The correctly sequenced recombinant expression vectors were named pU6-sgRNA1 (a recombinant expression vector containing sgRNA1) and pU6-sgRNA2 (a recombinant expression vector containing sgRNA2). The pU6-sgRNA1 map is shown below. Picture 1 As shown, the pU6-sgRNA2 map is as follows: Picture 2 As shown, and the sequencing peak diagrams of the two are as follows. Picture 3 As shown.

[0058] Example 2

[0059] Using the pU6-sgRNA recombinant expression plasmid prepared in Example 1, this example constructs... FAM50AThe gene knockout NIH / 3T3 and β-TC-6 cell lines specifically include the following steps:

[0060] (1) Culture of NIH / 3T3 cells and β-TC-6 cells.

[0061] Both NIH / 3T3 and β-TC-6 cells used in the experiment were purchased from ATCC and cultured in complete culture medium after revival. When the cell confluence reached 70%-80%, the cells were passaged, and cells that had been passaged twice were selected for subsequent experiments.

[0062] (2) Transfection of recombinant expression vector and screening of positive clones.

[0063] Cells were subjected to electroporation. FAM50A Gene knockout, specifically, involves the following steps:

[0064] (i) After adhering cells were digested with 0.25% Trypsin-EDTA, the cells were resuspended in Opti-MEM and the cell density was adjusted to 1×10⁻⁶. 6 250 μL;

[0065] (ii) Add 10 μg each of pU6-sgRNA1 and pU6-sgRNA2 recombinant expression plasmids to 250 μL of cell suspension, mix well and transfer to a 4 mm electroporation cuvette.

[0066] (iii) Electrotransfer is performed using an exponential waveform mode, with the following specific parameters:

[0067] NIH / 3T3 cells: Voltage 200-250 V, Capacitance 950 μF, Resistance ∞ Ω;

[0068] β-TC-6 cells: voltage 280-300 V, capacitance 950 μF, resistance ∞ Ω;

[0069] (iv) After electroporation, the cell suspension was seeded into high-glucose DMEM medium containing 10% fetal bovine serum (FBS) but without antibiotics and cultured for 24 h;

[0070] (v) After 24 h, the cells were digested with 0.25% Trypsin-EDTA to form a single-cell suspension. Single cells expressing brilliant blue fluorescent protein (BFP) were screened under a fluorescence microscope and then transferred into 96-well plates for cloning and amplification.

[0071] (3) FAM50A Identification of gene knockout monoclonal cell lines.

[0072] ① Genotype identification.

[0073] Collected and amplified monoclonal cell lines and extracted genomic DNA. Genotype identification primers were designed based on the target sites of sgRNA1 and sgRNA2 (see Table 5). The target gene sequences were amplified using PCR; the PCR reaction system and conditions are detailed in Tables 6 and 7. The PCR products were recovered by electrophoresis and then sequenced. The sequencing results are as follows: Picture 4 As shown, mutations have occurred in the genomes of both NIH / 3T3 and β-TC-6 positive monoclonal cells. Specifically, NIH / 3T3 positive monoclonal cells have a 1-base insertion at the sgRNA1 target site and a 6-base deletion at the sgRNA2 target site, while β-TC-6 positive monoclonal cells have 12-base deletions near both the sgRNA1 and sgRNA2 target sites.

[0074] Table 5 Primer sequences used for genotyping of sgRNA1 and sgRNA2 target sites.

[0075]

[0076] Table 6 PCR reaction system for genotype identification

[0077]

[0078] Table 7 PCR reaction conditions for genotype identification

[0079]

[0080] ②FAM50A protein expression analysis.

[0081] The expression of FAM50A protein in positive monoclonal cells was detected by Western blot. The specific procedure was as follows: wild-type cells (WT) and corresponding positive monoclonal cells (KO) were lysed with RIPA buffer containing protease inhibitors and phosphorylation inhibitors, respectively. The protein was separated by SDS-PAGE and wet-transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. Then, anti-FAM50A primary antibody (Prospertech, HU-412003) was added and incubated overnight at 4°C. The next day, the membrane was incubated with HRP-labeled goat anti-rabbit secondary antibody at room temperature for 1 h. ECL luminescent substrate was added for detection, and β-actin was used as an internal control.

[0082] Western blot results are as follows Picture 5 As shown, FAM50A protein expression was not detected in either NIH / 3T3 or β-TC-6 positive monoclonal cells, indicating that the PCR method was successfully constructed. FAM50A Gene knockout NIH / 3T3 and β-TC-6 cell lines can efficiently achieve... FAM50A Gene knockout.

[0083] In summary, the targeted mouse provided by this invention... FAM50A The sgRNA1 and sgRNA2 genes and their recombinant expression vectors can be used in combination to achieve efficient expression. FAM50A Gene knockout, and based on this, a gene was successfully constructed. FAM50A The knockout NIH / 3T3 and β-TC-6 cell lines are... FAM50A Gene function research provides stable and reliable experimental tools, offering important technical support for the study of the pathogenesis of FAM50A-related diseases and the development of potential treatment strategies.

[0084] It is important to note that although this invention employs a dual sgRNA design, it avoids the large-fragment genomic deletions commonly seen in traditional multi-sgRNA designs. Simultaneously, it effectively overcomes the incomplete knockout issues that may exist with single sgRNA designs, thus ensuring complete silencing of the FAM50A protein. The sgRNA pairs provided by this invention only induce small-fragment gene mutations (insertions or deletions) near the target site. Therefore, the technical solution of this invention effectively avoids widespread damage to gene structure, significantly improving the precision of gene editing and the biosafety of the constructed cell lines. Furthermore, this invention uses an electroporation combined with a non-integrative plasmid vector transient expression system to achieve CRISPR / Cas9 delivery, eliminating the need to screen for stable integration into cell lines. This effectively avoids foreign sequence residues caused by viral vector integration, thereby ensuring the genetic stability, safety, and reliability of the constructed gene-edited cell lines in subsequent research applications.

[0085] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A construction FAM50A The method for gene knockout mouse cell lines is characterized by, Includes the following steps: (1) The sequences of sgRNA1 and sgRNA2 were cloned into the plasmid pU6-sgRosa26-1_CBh-Cas9-T2A-BFP, respectively, to obtain the recombinant expression vector pU6-sgRNA1 containing the sgRNA1 sequence and the recombinant expression vector pU6-sgRNA2 containing the sgRNA2 sequence; wherein, the sequence of sgRNA1 is shown in SEQ ID NO.1, and the recognition site is located in mice. FAM50A The third exon region of the gene; the sequence of sgRNA2 is shown in SEQ ID NO.2, and the recognition site is located in mice. FAM50A The fourth exon region of the gene; (2) Electrotransfect the recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2 into the NIH / 3T3 cell line or the β-TC-6 cell line, and screen for positive single clones; (3) Genotyping and protein expression analysis were performed on the positive monoclonal antibodies, revealing that FAM50A protein expression was completely absent. FAM50A Gene knockout mouse cell lines.

2. The method according to claim 1, characterized in that, In step (1), the sgRNA1 sequence is integrated into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid using the primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the sgRNA2 sequence is integrated into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid using the primers shown in SEQ ID NO.5 and SEQ ID NO.

6.

3. The method according to claim 1 or 2, characterized in that, Step (2) Electroporation is performed using exponential wave mode; when the cell line is NIH / 3T3 cell line, the electroporation parameters are voltage 250 V, capacitance 950 μF, and resistance ∞ Ω; when the cell line is β-TC-6 cell line, the electroporation parameters are voltage 280 V, capacitance 950 μF, and resistance ∞ Ω.

4. The method according to claim 1 or 2, characterized in that, The genotyping in step (3) was performed using the primers shown in SEQ ID NO. 7-10.

5. The method constructed according to any one of claims 1-4 FAM50A Gene knockout mouse cell lines in FAM50A Applications in gene function research.

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