SgRNA of targeting mouse FAM50A gene and application of sgRNA of targeting mouse FAM50A gene

Abstract

The invention provides a pair of sgRNAs targeting a mouse FAM50A gene and application of the sgRNAs, and belongs to the technical field of gene editing, the sgRNAs comprise sgRNA1 and sgRNA2, recognition sites of the sgRNA1 and the sgRNA2 are located in the third exon region and the fourth exon region of the mouse FAM50A gene respectively, the sequence of the sgRNA1 is as shown in SEQ ID NO.1, and the sequence of the sgRNA2 is as shown in SEQ ID NO.2. Based on the sgRNA1 and sgRNA2 of the targeted FAM50A gene provided by the invention, an NIH / 3T3 cell line and a beta-TC-6 cell line of which the FAM50A gene is stably knocked out can be efficiently constructed by combining a CRISPR / Cas9 system, and a research tool and a material are provided for the functional research of the FAM50A gene and the development of related disease models.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, in particular to a pair of targeted mice FAM50A sgRNA targeting a gene and its application in constructing gene knockout cell lines. Background Art

[0002] FAM50A (Family with sequence similarity 50, member A), also known as Xchromosome-associated protein 5 ( XAP 5), a highly conserved gene linked to the X chromosome and widely expressed in various mammalian tissues. Studies have shown that abnormal FAM50A function is closely associated with genetic disorders 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 crucial for understanding its biological role and the pathogenesis of related diseases.

[0003] CRISPR / Cas9 gene editing technology relies on small guide RNA (sgRNA) to guide the Cas9 nuclease to specific DNA sequences. It achieves gene knockout or modification by inducing double-strand breaks and leveraging the cell's native non-homologous end joining (NHEJ) or homologous recombination (HDR) mechanisms. Notably, Cas9 and sgRNA require only a short period of expression and activity in cells, eliminating the need for long-term genomic integration to complete the editing process. The effectiveness of CRISPR / Cas9 technology is highly dependent on the design and selection of sgRNAs. Existing studies have shown that a single sgRNA design may not achieve complete knockout in some cases due to insufficient editing efficiency. Therefore, the combined use of multiple sgRNAs has become a common strategy to improve knockout efficiency. However, the combined use of multiple sgRNAs often results in large gene deletions or unintended genomic rearrangements, posing potential safety concerns in research and preclinical applications. Furthermore, while traditional integrative expression methods using viral vectors can effectively introduce the CRISPR / Cas9 system, they can easily lead to long-term retention of exogenous sequences in the cellular genome, compromising the genetic stability of cell models and the reliability of subsequent applications.

[0004] Currently, there is no study targeting mouse cell lines. FAM50A There are no public reports on efficient and specific sgRNA pairs for gene knockout and their corresponding recombinant expression vectors, nor is there a lack of cell lines that completely silence the FAM50A protein constructed through non-integration methods. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a pair of targeted mice FAM50A Gene sgRNA and its application in constructing gene knockout cell lines to solve the lack of reliable mouse-derived cell lines FAM50A Technical issues with gene knockout cell models.

[0006] In the first aspect, the present invention provides a pair of targeted mice FAM50A sgRNAs for genes, including: sgRNA1: 5'-GGCAAAAAAGGAGCAGTCAA-3' (SEQ ID NO. 1, located at FAM50A exon 3 of the gene); sgRNA2: 5'-AGAGATGGCTATGTACGAGG-3' (SEQ ID NO. 2, located at FAM50A exon 4 of the gene).

[0007] In a second aspect, the present invention provides a recombinant expression vector containing the above-mentioned sgRNA, the construction method of which is: The sequence of sgRNA1 was cloned into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid to obtain the recombinant expression vector pU6-sgRNA1; The sequence of sgRNA2 was cloned into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid to obtain the recombinant expression vector pU6-sgRNA2.

[0008] In some embodiments of the present invention, the site-directed mutagenesis method was used, and the sequences of sgRNA1 and sgRNA2 were respectively integrated into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid using the primers shown in SEQ ID NOs. 3-4 and SEQ ID NOs. 5-6, thereby obtaining recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2.

[0009] In the third aspect, the present invention provides the above-mentioned sgRNA and its recombinant expression vector for constructing FAM50A Application in gene knockout cell lines specifically includes the following steps: S1. Clone the sgRNA1 and sgRNA2 sequences 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; S2. Electroporate the recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2 into mouse cells and screen for positive monoclonal clones. S3. Identification of cells with complete loss of FAM50A protein expression FAM50A Knockout mouse cell lines.

[0010] In the above applications, mouse cells include NIH / 3T3 cell line and β-TC-6 cell line.

[0011] Preferably, in the above application, the primers shown in SEQ ID NO. 7-10 are used for identification.

[0012] In the fourth aspect, the method according to the present invention is constructed FAM50A Gene knockout cell lines also fall within the scope of protection of the present invention and can be used for FAM50A Fields such as gene function research, tumor mechanism research and drug development.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a pair of specific targeting FAM50A The sgRNA of the gene, combined with the CRISPR / Cas9 system, can achieve efficient and complete FAM50A Gene knockout. The sgRNA pairs designed by the present invention only cause small fragment insertions or deletions near the target site, without destroying the overall structure of the genome, avoiding the large genomic deletions common in traditional multi-sgRNA strategies, and have the advantages of precise targeting and high knockout efficiency. In addition, the present invention uses electrofection technology to achieve transient expression of the CRISPR / Cas9 system in host cells. Without screening cell lines that stably integrate exogenous plasmids, a method for constructing a cell model with complete deletion of FAM50A protein can be used, effectively avoiding the residual exogenous sequences caused by viral vector integration, thereby ensuring the genetic stability, safety and reliability of the constructed gene-edited cell line in subsequent research applications.

[0014] The present invention successfully knocked out the NIH / 3T3 cell line and the β-TC-6 cell line. FAM50A The experimental results show that the knockout is safe, reliable, and will not cause the deletion of large sections of genomic fragments. FAM50A The gene knockout method is very practical. FAM50A It provides effective methods and foundations for functional studies of genes and the pathogenesis of related diseases as well as the development of potential therapeutic strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0016] Picture 1 This is a map of the recombinant expression vector pU6-sgRNA1 in Example 1 of the present invention; Picture 2 This is a map of the recombinant expression vector pU6-sgRNA2 in Example 1 of the present invention; Picture 3 This is the sequencing peak diagram of the recombinant expression vectors pU6-sgRNA1 and pU6-sgRNA2 in Example 1 of the present invention; Picture 4 The genotype identification results of NIH / 3T3 and β-TC-6 positive monoclonal cells in Example 2 of the present invention; Picture 5 These are the results of Western blot detection of FAM50A protein in NIH / 3T3 and β-TC-6 positive monoclonal cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0017] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "include" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0019] Some specific examples are listed below. It should be noted that the examples 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 examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0020] Example 1 This example provides a pair of CRISPR / Cas9 gene knockout vectors. The construction process is as follows: (1) Targeting mice FAM50A sgRNA of the gene.

[0021] Based on the NCBI database FAM50A Gene sequence information (Gene ID: 108160), this example provides two targeted mice FAM50A sgRNA targeting genes, where sgRNA1 targets FAM50A sgRNA2 targets the exon 3 region of the gene FAM50A The specific sequence of the 4th exon region of the gene is as follows: sgRNA1: 5'-GGCAAAAAAGGAGCAGTCAA-3' (SEQ ID NO.1); sgRNA2: 5'-AGAGATGGCTATGTACGAGG-3' (SEQ ID NO.2).

[0022] (2) Construction of pU6-sgRNA recombinant expression vector.

[0023] 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 a recombinant expression vector. The process is as follows: ① Design and synthesize primers for site-directed mutagenesis, as shown in Table 1.

[0024] Table 1 Point mutation primer sequences corresponding to sgRNA1 and sgRNA2

[0025] ②Preparation of linearized vector.

[0026] The vector was amplified by inverse PCR using Phanta Max Super-Fidelity DNA Polymerase (Vazyme #P505), wherein the reaction system is shown in Table 2 and the reaction conditions are shown in Table 3.

[0027] Table 2 Reverse PCR reaction system

[0028] Table 3 Reaction conditions for inverse PCR

[0029] ③ Amplification product DPN I digestion.

[0030] Add 1 μl to the amplified product from step ② DPN I, incubate at 37°C for 2 h to digest and remove the methylated template plasmid in the product.

[0031] ④ In vitro recombination of linearized vector.

[0032] The digested linearized vector was circularized and recombined in vitro using Exnase II catalytic enzyme. The reaction system is shown in Table 4.

[0033] Table 4 In vitro cyclization reaction system of linearized vector

[0034] After the reaction system was prepared, it was kept at 37°C for 30 min and then immediately cooled on ice.

[0035] ⑤Transformation of recombinant expression plasmid.

[0036] Take the DH5α competent cells stored at -80℃ and thaw them on ice; then take 100 μl of competent cells, add 10 μl of the recombination reaction ligation product obtained in step ④, mix well and let it stand on ice for 30 minutes; heat shock in a 42℃ water bath for 45 seconds, then immediately place it on ice to cool for 3 minutes; add 900 μl of LB medium without antibiotics and incubate at 37℃ on a shaker for 1 hour (speed 200-250 rpm); spread LB plates containing AMP (100 μg / mL) and culture at 37℃ overnight; pick positive clones and sequence them after incubation at 37℃ on a shaker overnight. The correctly sequenced bacterial solution samples were preserved and plasmids were extracted. The correctly sequenced recombinant expression vectors were named: pU6-sgRNA1 (recombinant expression vector containing sgRNA1) and pU6-sgRNA2 (recombinant expression vector containing sgRNA2). The map of pU6-sgRNA1 is shown below. Picture 1 As shown, the map of pU6-sgRNA2 is as follows Picture 2 As shown, and the sequencing peak diagrams of the two are as follows Picture 3 shown.

[0037] Example 2 Using the pU6-sgRNA recombinant expression plasmid prepared in Example 1, this example constructed FAM50A Gene knockout NIH / 3T3 cell line and β-TC-6 cell line, specifically including the following steps: (1) Culture of NIH / 3T3 cells and β-TC-6 cells.

[0038] The NIH / 3T3 cells and β-TC-6 cells used in the experiment were purchased from ATCC and cultured in complete medium after recovery. Cells were passaged when the confluence reached 70%-80%, and cells after two passages were selected for subsequent experiments.

[0039] (2) Transfection of recombinant expression vector and screening of positive clones.

[0040] Cells were electroporated using FAM50A Gene knockout, the specific operation is: (i) After the adherent cells were digested with 0.25% Trypsin-EDTA, they were resuspended in Opti-MEM and the cell density was adjusted to 1×10 6 / 250 μL; (ii) Add 10 μg 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; (iii) Electrotransfection was performed using an exponential waveform mode with the following parameters: NIH / 3T3 cells: voltage 200-250 V, capacitance 950 μF, resistance ∞ Ω; β-TC-6 cells: voltage 280-300 V, capacitance 950 μF, resistance ∞ Ω; (iv) After electroporation, the cell suspension was inoculated into high-glucose DMEM medium containing 10% fetal bovine serum (FBS) but without antibiotics and cultured for 24 h; (v) After 24 h, cells were digested with 0.25% Trypsin-EDTA to obtain a single-cell suspension. Single cells expressing brilliant blue fluorescent protein (BFP) were screened under a fluorescence microscope and transferred into a 96-well plate for clonal expansion.

[0041] (3) FAM50A Identification of knockout monoclonal cell lines.

[0042] ① Genotype identification.

[0043] The monoclonal cell lines were collected and amplified, and genomic DNA was extracted. Genotype identification primers were designed according to the target sites of sgRNA1 and sgRNA2 (see Table 5 for details). The target gene sequence was amplified by PCR. The PCR reaction system and conditions are detailed in Tables 6 and 7. The PCR products were recovered by electrophoresis and sequenced. The sequencing results were as follows: Picture 4 As shown, the genomes of both NIH / 3T3 and β-TC-6 positive monoclonal cells were mutated; specifically, NIH / 3T3 positive monoclonal cells had 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 had a 12-base deletion near both the sgRNA1 and sgRNA2 target sites.

[0044] Table 5 Primer sequences used for genotyping of sgRNA1 and sgRNA2 target sites

[0045] Table 6 Genotype identification PCR reaction system

[0046] Table 7 PCR reaction conditions for genotyping

[0047] ②FAM50A protein expression analysis.

[0048] Western blot was used to detect the expression of FAM50A protein in positive monoclonal cells. The specific procedures were as follows: wild-type cells (WT) and corresponding positive monoclonal cells (KO) were lysed with RIPA buffer containing protease inhibitors and phosphorylation inhibitors, proteins were separated by SDS-PAGE, and the proteins were wet-transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 h, and then anti-FAM50A primary antibody (Prospertech, HU-412003) was added. The membranes were incubated at 4°C overnight. The next day, HRP-labeled goat anti-rabbit secondary antibody was incubated at room temperature for 1 h. ECL luminescent substrate was added for detection, and β-actin was used as an internal control.

[0049] Western blot results are as follows Picture 5 As shown in Figure 2, the expression of FAM50A protein could not be detected in NIH / 3T3 and β-TC-6 positive monoclonal cells, indicating that the FAM50A protein was successfully constructed. FAM50A Gene knockout NIH / 3T3 cell line and β-TC-6 cell line can achieve high efficiency FAM50A Gene knockout.

[0050] In summary, the targeted mice provided by the present invention FAM50A The sgRNA1 and sgRNA2 of the gene and their recombinant expression vector can be combined to achieve efficient FAM50A gene knockout, and based on this, we successfully constructed FAM50A Knockout NIH / 3T3 cell line and β-TC-6 cell line, FAM50A It provides a stable and reliable experimental tool for gene function research and offers important technical support for the study of the pathogenesis of FAM50A-related diseases and the development of potential treatment strategies.

[0051] It is important to note that despite employing a dual-sgRNA design, the present invention avoids the large-scale genomic deletions commonly seen with traditional multiple-sgRNA interactions. Furthermore, it effectively overcomes the incomplete knockout problem that can occur with a single-sgRNA design, thereby ensuring complete silencing of the FAM50A protein. The sgRNA pairs provided by the present invention only induce small gene mutations (insertions or deletions) near the target site. Therefore, the technical solution of the present invention effectively avoids extensive damage to the gene structure, significantly improving the accuracy of gene editing and the biosafety of the constructed cell lines. Furthermore, the present invention utilizes a transient expression system combining electrofection with a non-integrating plasmid vector to achieve CRISPR / Cas9 introduction, eliminating the need to screen for stably integrated cell lines and effectively avoiding the presence of exogenous sequences caused by viral vector integration, thereby ensuring the genetic stability, safety, and reliability of the constructed gene-edited cell lines in subsequent research applications.

[0052] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A pair of targeted mice FAM50A The sgRNA of a gene is characterized by Including sgRNA1 and sgRNA2, the recognition sites of sgRNA1 and sgRNA2 are respectively located in mouse FAM50A The 3rd and 4th exon regions of the gene, the sequence of sgRNA1 is shown as SEQ ID NO.1, and the sequence of sgRNA2 is shown as SEQ ID NO.

2.

2. The sgRNA according to claim 1 or the recombinant expression vector containing the sgRNA is constructed FAM50A Application in gene knockout cell lines, including NIH / 3T3 cell line and β-TC-6 cell line.

3. The use according to claim 2, characterized in that The recombinant expression vector is obtained by cloning the sequence of the sgRNA1 and / or sgRNA2 into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid.

4. The use according to claim 3, characterized in that Using the site-directed mutagenesis method, the sequences of sgRNA1 and sgRNA2 were integrated into the pU6-sgRosa26-1_CBh-Cas9-T2A-BFP plasmid using the primers shown in SEQ ID NO.3-4 and the primers shown in SEQ ID NO.5-6, respectively.

5. A method according to any one of claims 2 to 4. FAM50A Knockout mouse cell lines FAM50A Application in gene function research, tumor mechanism research and drug development tools.

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