Application of chloroquine in improving gene editing efficiency

Abstract

This invention discloses the application of chloroquine in improving gene editing efficiency. Specifically, it provides a novel use for chloroquine: enhancing the gene editing efficiency of gene editing reagents on recipient biological materials. The gene editing reagent is a gene editing tool plasmid. No prior art studies have combined chloroquine with gene editing efficiency. The inventors of this invention have discovered that treating recipient biological materials with chloroquine before transfecting them with gene editing reagents can significantly improve gene editing efficiency. This invention is simple to operate, widely applicable, and opens up a new avenue for improving editing efficiency by introducing exogenous small molecules rather than directly modifying gene editing tools.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a new use of chloroquine, specifically its application in improving gene editing efficiency. Background Technology

[0002] CRISPR-Cas9 technology originates from an adaptive immune system in bacteria and archaea. Its core editing principle lies in using a programmable single-stranded guide RNA (sgRNA) to precisely guide the Cas9 nuclease to a specific target site in the genome through base complementarity pairing. The Cas9 protein then cleaves the DNA double strand at that site, creating a double-strand break. The cell then initiates DNA repair mechanisms to repair this break, mainly through two pathways: joining non-homologous ends of gene knockout insertions or deletions; and homologous recombination repair through precise gene knock-in or correction when an exogenous repair template is available.

[0003] CRISPR-Cas9 technology has revolutionized gene editing due to its simple design, low cost, and high efficiency. However, its editing efficiency still faces multiple challenges in practical applications: the selection of endogenous cellular repair pathways, chromatin state (such as poor accessibility of highly condensed heterochromatin regions), epigenetic modifications of target sites, and cell delivery efficiency all constitute intracellular barriers affecting editing efficiency, thus reducing it. These efficiency-related issues collectively represent key scientific bottlenecks that must be continuously optimized and overcome in the process of CRISPR-Cas9 technology moving towards clinical treatment and wider application.

[0004] Chloroquine, a classic antimalarial drug, primarily works through its weakly alkaline properties by entering acidic organelles (such as lysosomes and endosomes), raising their pH levels, and thus interfering with various cellular processes. In malaria infection, chloroquine inhibits the degradation of hemoglobin in the food vacuoles of Plasmodium, leading to the accumulation of toxic heme and killing the parasite. During autophagy, it inhibits autophagic flux and substrate degradation by disrupting the acidic environment of lysosomes. In addition, chloroquine can also affect endosome transport and antigen presentation. Summary of the Invention

[0005] The purpose of this invention is to provide the application of chloroquine in improving gene editing efficiency.

[0006] This invention provides a new use for chloroquine, namely, its application in improving gene editing efficiency.

[0007] Specifically, this invention provides the application of chloroquine in improving the gene editing efficiency of gene editing reagents.

[0008] Specifically, this invention provides the application of chloroquine in improving the gene editing efficiency of gene editing reagents on recipient biomaterials.

[0009] This invention provides the application of chloroquine in gene editing.

[0010] This invention provides the use of chloroquine in the preparation of kits for gene editing.

[0011] This invention provides the application of chloroquine and gene editing reagents in gene editing.

[0012] Specifically, chloroquine enhances the gene-editing efficiency of gene-editing reagents.

[0013] Specifically, chloroquine enhances the gene editing efficiency of gene editing agents on recipient biomaterials.

[0014] This invention provides the application of chloroquine and gene editing reagents in the preparation of kits for gene editing.

[0015] This invention provides a kit for gene editing, comprising chloroquine and gene editing reagents.

[0016] The present invention also provides a method for improving gene editing efficiency, comprising the following steps: treating the recipient biological material with chloroquine before transfecting the recipient biological material with gene editing reagent.

[0017] Specifically, any of the above-described receptor biomaterials can be cells. Specifically, any of the above-described cells can be animal cells. Specifically, any of the above-described cells can be mammalian cells. Specifically, any of the above-described cells can be human cells. For example, any of the above-described cells can be HEK293T cells.

[0018] In this application, the concentration of chloroquine used is 7.5-15 µM. In this application, the concentration of chloroquine used is 7.5-10 µM or 10-15 µM. In this application, the concentration of chloroquine used is 7.5 µM, 10 µM, or 15 µM. In this application, the working concentration of chloroquine is 7.5-15 µM. In this application, the working concentration of chloroquine is 7.5-10 µM or 10-15 µM. In this application, the working concentration of chloroquine is 7.5 µM, 10 µM, or 15 µM.

[0019] In the method described above, when treating the recipient biological material with chloroquine, the concentration of chloroquine is 7.5-15 µM. Alternatively, the concentration of chloroquine used in the method described above is 7.5-10 µM or 10-15 µM. Alternatively, the concentration of chloroquine used in the method described above is 7.5 µM, 10 µM, or 15 µM.

[0020] In the method described above, the recipient biological material is treated with chloroquine for 4-6 hours. In another method, the recipient biological material is treated with chloroquine for 5 hours.

[0021] The gene editing reagents described above are gene editing reagents delivered in the form of plasmid DNA, gene editing reagents delivered in the form of mRNA, or gene editing reagents in the form of ribonucleoprotein complexes.

[0022] Specifically, the gene editing reagent is a gene editing tool plasmid. Specifically, the gene editing tool plasmid is a CRISPR-Cas9 gene editing tool plasmid. Specifically, the gene editing tool plasmid is a plasmid expressing Cas9 nuclease and sgRNA; the sgRNA is an sgRNA targeting the target gene. Specifically, the gene editing tool plasmid is a plasmid expressing Cas9 nuclease and a plasmid expressing sgRNA; the sgRNA is an sgRNA targeting the target gene. As an example, the gene editing tool plasmid is a plasmid obtained by modifying a recombinant plasmid; the full sequence of the recombinant plasmid is shown in SEQ ID NO: 1; the modification refers to replacing the DNA segment shown in positions 251-270 of SEQ ID NO: 1 with a DNA segment encoding the target sequence binding region of the target gene in the sgRNA.

[0023] As an example, the target gene is the human EMX1 gene.

[0024] As an example, the gene editing tool plasmid is a recombinant plasmid with a full sequence as shown in SEQ ID NO: 1.

[0025] No studies in the prior art have combined chloroquine with gene editing efficiency, and there are no reports on the effect of chloroquine on Cas9 editing efficiency. The inventors of this invention have discovered that treating cells to be transfected with chloroquine before transfecting them with CRISPR / Cas9 tool plasmids can significantly improve the gene editing efficiency of CRISPR / Cas9 tools for target genes. This invention is simple to operate and widely applicable, opening up a new avenue for improving editing efficiency by introducing exogenous small molecules rather than directly modifying gene editing tools. This strategy provides an innovative solution for more efficient and safer gene editing technology applications. Attached Figure Description

[0026] Figure 1 The image shows the results of Example 2 (the test compound was chloroquine).

[0027] Figure 2 The result diagram is from Example 2 (the test compound is SURAMIN). Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. HEK293T cells: ATCC, catalog number CRL-3216. Lipofectamine™ 3000 transfection reagent (Invitrogen™): Thermo Fisher Scientific, catalog number L3000075; Lipofectamine™ 3000 transfection reagent is provided in two components: Lipofectamine 3000 reagent and P3000™ reagent. Opti-MEM™ I serum-reduced medium (Gibco™): Thermo Fisher Scientific, catalog number 31985070. Complete medium: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Unless otherwise specified, the cell culture conditions were 37°C and 5% CO2. Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged. **** indicates p < 0.0001, indicating a significant difference; * indicates p < 0.05, indicating a significant difference; ns indicates that the significance level was not reached.

[0030] 10mM chloroquine solution (solvent is DMSO): MCE, product catalog number HY-17589A; the structural formula of chloroquine is shown in formula (Ⅰ), CAS number 54-05-7.

[0031] Equation (Ⅰ).

[0032] 10mM SURAMIN solution (solvent is DMSO): MCE, product catalog number HY-B0879A; the structural formula of SURAMIN (Suramin sodium salt) is shown in formula (II), CAS number is 129-46-4.

[0033] Formula (II).

[0034] This example uses the human EMX1 gene as an example to verify the effect of chloroquine on gene editing efficiency. The human EMX1 gene is located on chromosome 2, and its sequence can be found at positions 26231-44933 in GenBank: AC012366.10 (23-JUL-2016). To facilitate the illustration of the target sequence of sgRNA, a partial segment of the human EMX1 gene is described in the specification, as shown in SEQ ID NO: 2.

[0035] Example 1: Construction of recombinant plasmids The recombinant plasmid is a circular plasmid formed from double-stranded DNA molecules. The full sequence of the recombinant plasmid is shown in SEQ ID NO: 1. In SEQ ID NO: 1, nucleotides 251-346 encode sgRNA (nucleotides 251-270 encode the target sequence binding region of sgRNA, and nucleotides 271-346 encode the backbone region of sgRNA), and nucleotides 2310-6410 encode the Cas9 protein. The target sequence of sgRNA is (boldly marked PAM): GTCACCTCCAATGACTAGGGTGG.

[0036] Example 2: Validation of the compound's effect on gene editing The test compound is either SURAMIN or chloroquine.

[0037] The test compound solution was either 10 mM SURAMIN solution or 10 mM chloroquine solution.

[0038] 1. Take HEK293T cells and suspend them in complete culture medium to make the cell density 1.8 × 10⁻⁶. 6 Cells / mL is called cell suspension.

[0039] 2. Take a 12-well plate, inoculate each well with 0.1 mL of the cell suspension prepared in step 1 and 1 mL of complete culture medium, and then incubate for 24 hours.

[0040] 3. After completing step 2, add samples to the groups and then incubate for 5 hours.

[0041] The grouping and sampling method is as follows: Experimental group 1 (Cas9+15μM): Add the test compound solution to make the concentration of the test compound in the system 15μM; Experimental group 2 (Cas9+10μM): Add the test compound solution to make the concentration of the test compound in the system 10μM; Experimental group 3 (Cas9 + 7.5 μM): Add the test compound solution to make the concentration of the test compound in the system 7.5 μM; Control group 1 (Cas9): No samples were added; Control group 2 (WT): No samples were added; Each group has 3 duplicate holes.

[0042] 4. After completing step 3, take the 12-well plate and add the transfection complex to experimental group 1, experimental group 2, experimental group 3 and control group 1. No sample is added to control group 2. Then, incubate for 24 hours.

[0043] Preparation method of transfection complex for one well: ① Mix 75 μl of Opti-MEM™ I serum-reduced medium and 2.5 μl of Lipofectamine 3000 reagent, and let stand at room temperature for 5 min; ② Mix 75 μl of Opti-MEM™ I serum-reduced medium, 2 μg of recombinant plasmid prepared in Example 1 and 2 μl of P3000™ reagent, and let stand at room temperature for 5 min; ③ Mix the liquid phase from step ① and the liquid phase from step ②, and let stand at room temperature for 10-15 min.

[0044] 5. After completing step 4, discard the supernatant, add complete culture medium, and incubate for 24 hours.

[0045] 6. After completing step 5, collect cells by pressing the wells.

[0046] 7. Take the cells obtained in step 6 and extract genomic DNA. Using the genomic DNA as a template, perform PCR amplification using a primer pair consisting of DAPI-32-F and DAPI-32-R. Then collect the amplification products and perform high-throughput sequencing. Calculate the gene editing efficiency of each well based on the sequencing results.

[0047] DAPI-32-F: 5'-ctttccctacacgacgctcttccgatctgtccgagcagaagaagaagg-3'; DAPI-32-R: 5'-gttccttggcacccgagaattccatgcttgtccctctgtcaatg-3'.

[0048] The method for calculating gene editing efficiency is as follows: If the amplification products of the cells in the well undergo high-throughput sequencing and yield 40,000 sequences, of which 10,000 sequences are wild-type sequences (wild-type sequences are those containing the region shown in SEQ ID NO: 2, 353-621), and 30,000 sequences are non-wild-type sequences (non-wild-type sequences are those not containing the region shown in SEQ ID NO: 2, 353-621), then the gene editing efficiency of the well is 75%.

[0049] The result is the average of the three replicates.

[0050] When the test compound is chloroquine, the results are shown in [the table below]. Figure 1 The gene editing efficiency of control group 1 was 38%. The gene editing efficiency of experimental group 1 was 42%, a 1.11-fold increase compared to control group 1. The gene editing efficiency of experimental group 2 was 42%, a 1.11-fold increase compared to control group 1. The gene editing efficiency of experimental group 3 was 54%, a 1.42-fold increase compared to control group 1. The results indicate that chloroquine can significantly improve the editing efficiency of the Cas9 editing system at three different concentrations, with the optimal effect observed at a concentration of 7.5 μM.

[0051] When the test compound is SURAMIN, the results are shown in [the table below]. Figure 2 The gene editing efficiency of control group 1 was 44.7%. The gene editing efficiency of experimental group 1 was 37.8%, decreasing to 0.85 of control group 1. The gene editing efficiency of experimental group 2 was 38.2%, decreasing to 0.85 of control group 1. The gene editing efficiency of experimental group 3 was not significantly different from that of control group 1. The results indicate that SURAMIN cannot improve the editing efficiency of the Cas9 editing system.

[0052] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of chloroquine in improving gene editing efficiency.

2. Application of chloroquine in improving the gene editing efficiency of gene editing reagents.

3. The application of chloroquine in gene editing.

4. Application of chloroquine in the preparation of kits for gene editing.

5. Application of chloroquine and gene editing reagents in gene editing.

6. Application of chloroquine and gene editing reagents in the preparation of kits for gene editing.

7. Kits for gene editing, which include chloroquine and gene editing reagents.

8. A method for improving gene editing efficiency, comprising the following steps: treating the recipient biological material with chloroquine before transfecting it with a gene editing reagent.

9. The method as described in claim 8, characterized in that: The receptor biomaterial is a cell.

10. The method as described in claim 8 or 9, characterized in that: When treating recipient biomaterials with chloroquine, the concentration of chloroquine is 7.5-15 µM.

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