A method for improving gene editing efficiency by adding crotonic acid

Abstract

The present invention provides a method for improving gene editing efficiency by adding crotonic acid, which relates to the technical field of plant genetic engineering, and comprises the following steps: step 1: preparing a PE protein expression vector; step 2: constructing a pegRNA expression vector; step 3: adding crotonic acid to a gene to be edited, and culturing for 48 hours; step 4: transfecting and introducing the PE protein expression vector prepared in step 1 and the pegRNA expression vector constructed in step 2 into the gene to be edited to which the crotonic acid is added, so that gene editing occurs at a genomic DNA target site; the present invention uses crotonic acid for improving crotonylation of cell histones, and when a gene editing system is used for gene editing, crotonic acid is added to treat cells, thereby improving base substitution in the gene editing system, obtaining higher gene editing efficiency, solving the problem of low activity in precise site-specific gene editing, and providing a new method for efficient use of gene editors in mammalian cells.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a method for improving gene editing efficiency by adding crotonic acid. Background Art

[0002] As an important research field in life sciences, the development and application of gene editing technology has brought the genetic modification of organisms to an unprecedented depth and breadth. By introducing genetic modifications such as replacement, insertion and deletion into the genome, agricultural traits can be improved to accelerate the improvement and breeding of plants and animals, disease models can be constructed to explore the occurrence and progression of diseases and drug screening, and endogenous pathogenic mutations can be repaired or modified to achieve the purpose of gene therapy.

[0003] CPISPR-Cas9-mediated gene editing is mainly generated through DSBs, so there is a relatively large possibility of causing problems such as excess byproducts, uncontrolled insertion or deletion of gene transposition, and chromosomal abnormalities, which limit its ability to play a more important role.

[0004] Single-base editors can achieve specific base transversions or conversions (CBE, ABE, GBE) without relying on DSBs, but cannot achieve arbitrary conversions, deletions, and insertions between the 12 bases.

[0005] Prime editing system (PE) is a precise gene editing technology that can programmatically install substitutions, insertions, and deletions in cells and animals without double-stranded DNA breaks (DSBs). PE is a new gene editing method that can not only achieve arbitrary conversion between 12 single bases, but also insert and delete large fragments up to 10kb. However, the editing efficiency of the PE editing system in various cell lines is currently very low and unstable, which greatly limits its application. Therefore, how to improve the efficiency of PE is a difficult problem that needs to be solved. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for improving gene editing efficiency by adding crotonic acid. When using a gene editing system for gene editing, crotonic acid is added to treat cells to improve the openness of chromatin, promote the binding of PE to chromatin, and improve the efficiency of PE, thereby improving the editing efficiency of PE and the activity of the gene editing system to obtain higher gene editing efficiency, as follows:

[0007] A method for improving gene editing efficiency by adding crotonic acid comprises the following steps:

[0008] Step 1: Prepare PE protein expression vector;

[0009] Step 2: construct pegRNA expression vector;

[0010] Step 3: Add crotonic acid to the gene to be edited and culture for 48 hours;

[0011] Step 4: Transfect the PE protein expression vector prepared in step 1 and the pegRNA expression vector constructed in step 2 into the gene to be edited with crotonic acid added, so that gene editing occurs at the genomic DNA target site.

[0012] Moreover, in step 2, the pegRNA expression vector is used to specifically target the genomic DNA target site.

[0013] Moreover, in step 3, the gene to be edited is the IGF2 gene.

[0014] The IGF2 gene encodes insulin-like growth factor 2 (IGF2), which is a polypeptide that promotes cell division, participates in growth and development, and affects skeletal muscle mass and fat deposition. The IGF2 gene is highly conserved, and the destruction of the repressor zinc finger protein 6 (Zinc finger bed domaincontaining, ZBED6) binding site on intron 3 will cause the expression of IGF2 in skeletal muscle to increase, thereby affecting muscle growth and fat deposition.

[0015] Furthermore, the target site of the DNA is a mutation from G to A in intron 3 of the IGF2 gene.

[0016] Moreover, in step 3, the concentration of crotonic acid is 20 mM.

[0017] Moreover, in the step 2, the pegRNA expression vector is constructed as follows: a pegRNA fragment is obtained by annealing a single-stranded DNA, and the pegRNA fragment is connected to the expression vector U6-pegRNA-GG-acceptor to obtain a pegRNA expression vector;

[0018] The nucleotide sequence of the pegRNA is shown in SEQ ID NO.1.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Histone crotonylation (Kcr) modification is a modification produced by histone crotonyl transferase transferring a crotonyl group to a lysine residue using crotonyl-CoA as a substrate. This modification can neutralize the positive charge of the residue. The loss of positive charge on the histone lysine residue weakens DNA interaction, thereby reducing the compactness of chromatin, which is beneficial to transcriptional activation. Crotonic acid can significantly increase the overall histone crotonylation (Kcr) level and the level of histone Kcr in cells. Histone crotonylation modification participates in gene regulation. The present invention uses crotonic acid to increase cellular histone crotonylation (Kcr). When using a gene editing system for gene editing, crotonic acid is added to treat the cells, thereby improving base substitution in the gene editing system, obtaining higher gene editing efficiency, solving the problem of low activity in precise site-specific gene editing, and providing a new method for the efficient use of gene editors in mammalian cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To investigate the effect of adding crotonic acid on cell morphology;

[0022] Figure 2 is the effect of crotonic acid on crotonylation, Figure 2 a is a Western blotting image; Figure 2 b Protein gray value analysis chart;

[0023] Figure 3 The effect of adding crotonic acid on cell proliferation;

[0024] Figure 4 This is a picture showing that the editing efficiency is improved by adding crotonic acid;

[0025] Figure 5 This is the Sanger sequencing map. DETAILED DESCRIPTION

[0026] Example 1 Construction of PE protein expression vector and pegRNA expression vector

[0027] Step 1: Purchase PE protein expression vector Addgene (#132776) from Addgene;

[0028] Step 2: Construction of pegRNA expression vector: connect the synthesized pegRNA fragment into the expression vector U6-pegRNA-GG-acceptor (BsaAI and BsaBI double-digested backbone) to obtain the pegRNA expression vector;

[0029] The sequence of the pegRNA is shown in SEQ ID NO.1.

[0030] Example 2 Cultivation of bovine fetal fibroblasts

[0031] Take out the cryopreserved tube of bovine fetal fibroblasts from liquid nitrogen, immediately put it in a 37℃ water bath, shake it gently, and after the liquid is completely melted, centrifuge it at 1500rpm for 5min; discard the supernatant, add 1ml cell culture medium to resuspend the cells, and then transfer them to a 10cm culture dish containing 7ml culture medium and shake it gently back and forth and left and right to evenly distribute the cells in the culture dish; mark the cell type, date, name of the culturer, etc., and culture it in a 37℃, 5% CO2 incubator. After the cells adhere to the wall and grow full, subsequent operations can be carried out.

[0032] Preparation of complete culture medium: DMEM + 10% FBS (fetal bovine serum).

[0033] Example 3 Effect of adding crotonic acid on cell activity

[0034] 1. Plating of bovine fetal fibroblasts: Count the cells and inoculate 1.2×10 6 of cells.

[0035] 2. After the cells grew for 24 hours, a control group (CON) was set up and DMSO was added. Crotonic acid was added at concentrations of 10Mm, 20mM, 30Mm, 40Mm, and 50mM, respectively, and cultured for 24 hours.

[0036] 3. Test cell viability of bovine fetal fibroblasts:

[0037] a. After culturing the cells for 24 hours, digest and resuspend the cells and plate them into a 96-well plate at a density of 2,000 cells per well, with three replicates per group;

[0038] b. Incubate the culture plate in an incubator for 12 hours;

[0039] c. Add 10 μL of CCK-8 solution to each well (be careful not to create bubbles);

[0040] d. Place the culture plate in an incubator and incubate for 1-4 hours;

[0041] f. Measure the absorbance at 450 nm using an enzyme-labeled instrument. Measure the absorbance at 24h, 48h, 72h, and 96h of culture. Figure 1 , Figure 2 , Figure 3 ).

[0042] from Figure 1 It can be seen that different concentrations of crotonic acid have a significant effect on the morphology of fibroblasts. Under normal circumstances, fibroblasts usually appear spindle-shaped or flat star-shaped with extended protrusions. However, when crotonic acid is added, the morphology of the cells begins to change.

[0043] One change is that the cells shrink, changing from their original spindle shape to a round shape. This change in shape may be because crotonic acid has a toxic effect on the cells, destroying their normal structure and osmotic pressure balance, causing the cells to be unable to maintain their original shape.

[0044] Another change is that the protrusions of the cells retract, making the overall shape look more thin and strip-like. This change may be a response of the cells to crotonic acid stress. Since crotonic acid changes the chemical environment of the cells, the internal skeletal structure of the cells may be adjusted, thereby affecting and changing the appearance of the cells.

[0045] As the concentration of crotonic acid increased, the state of the cells showed a trend of gradual deterioration, which indicates that the toxic effect of crotonic acid on cells is closely related to its concentration. The higher the concentration, the greater the damage to the cells.

[0046] from Figure 2 It can be observed that with the gradual increase of the concentration of crotonic acid, the level of crotonylation modification also increased significantly; although according to Figure 1 From the results, we know that high concentrations of crotonic acid will cause the cell state to gradually deteriorate, but on the other hand, the level of crotonylation modification increases with the increase of crotonic acid concentration; to verify this trend, we used Western blotting for further analysis. The experimental results showed that with the increase of crotonic acid concentration, the color of the target protein band gradually deepened, indicating that the degree of crotonylation modification was constantly increasing; in order to quantify this change more accurately, we also used ImageJ software to analyze the color depth (i.e., grayscale value) of the target band; the analysis results showed that the darker the band color, the higher the corresponding crotonylation modification level; although high concentrations of crotonic acid have a negative impact on the cell state, it also promotes the increase of crotonylation modification level.

[0047] Figure 3 It can be seen that after adding different concentrations of crotonic acid, the proliferation of cells will slow down as the concentration increases. After adding crotonic acid, the cell proliferation at 10mM and 20mM is similar to that at 0mM. Starting from 30mM, the cell proliferation becomes slower and slower.

[0048] In summary, combined with Figure 1 , 23. It can be seen that although the higher the concentration of crotonic acid, the higher the crotonyl modification. However, the higher the concentration (30mM, 40mM, 50mM), the worse the cell morphology and proliferation; among 10mM and 20mM, although the proliferation rate of 10mM is slightly faster than that of 20mM, the degree of crotonyl modification is higher at 20mM concentration, so it can be determined that the best concentration of crotonic acid is 20mM.

[0049] Example 4 Adding crotonic acid to improve the editing efficiency of the PE system

[0050] 1. Plating of bovine fetal fibroblasts: Count the cells and inoculate 1.2×10 6 of cells.

[0051] 2. After the cells have grown for 24 h, the plasmid PE2 expression vector (3.75 μg) and pegRNA expression vector (1.25 μg) are transfected using the transfection reagent ExFect (Vazyme, #T101).

[0052] 3. Transfection method of bovine fetal fibroblasts: Add 200μl opti-MEM serum-free medium to a 1.5ml sterile centrifuge tube, add 10μl ExFect, and mix gently with a pipette. Add 200μlopti-MEM to another 1.5ml sterile centrifuge tube, add PE protein expression vector plasmid (3.75μg) and pegRNA expression vector plasmid (1.25μg), and mix gently with a pipette; add ExFect-opti-MEM dropwise to DNA-opti-MEM, mix gently with a pipette, blow and mix well, let stand at room temperature for 15-20min, and slowly add 10% FBS complete medium to a 6-well plate along the side wall of the culture dish for transfection.

[0053] 4. Before transfection, crotonic acid with a final concentration of 20 mM was added to the experimental group, and the corresponding volume of DMSO was added to the control group (CON) and cultured for 24 h.

[0054] 5. Collect cells 48 hours after transfection, select with puromycin for 3 days, collect cells again, extract DNA genome, perform PCR amplification, and compare the improvement of editing efficiency of PE editing system after adding crotonic acid by high-throughput sequencing. The results are shown in Figure 4 , Figure 5 .

[0055] PCR system:

[0056] 1 μl of DNA template, 1 μl of upstream primer (IGF2-F), 1 μl of downstream primer (IGF2-R), 10 μl of SYBR green mastermix (TaKaRa, China) and 7 μl of RNase-free H2O, a total of 20 μl.

[0057] The nucleotide sequence of IGF2-F is shown in SEQ ID NO.2;

[0058] The nucleotide sequence of IGF2-R is shown in SEQ ID NO.3.

[0059] PCR reaction system:

[0060] Pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 57°C for 30 s, extension at 72°C for 15 s, complete extension for 2 min, and the number of cycles was 30×.

[0061] The PCR product library was purified using the Agencourt AMPure XP Nucleic Acid Purification Kit (Beckman Coulter), and the purified PCR product library was mixed for high-throughput sequencing.

[0062] from Figure 4 It can be seen that in the absence of exogenous interference and after adding exogenous crotonic acid, the editing efficiency was increased by 0.3% after editing with primeediting.

[0063] Figure 5 It can be seen that the base G at the intron 3 of IGF2 to be edited mutated to A after prime editing.

Claims

1. A method for improving gene editing efficiency by adding crotonic acid, which is not for the purpose of disease treatment, characterized in that: The steps include: Step 1: Prepare PE protein expression vector; Step 2: constructing a pegRNA expression vector; the pegRNA expression vector is used to identify and specifically target genomic DNA target sites; Step 3: adding crotonic acid to the gene to be edited and culturing for 48 hours; the gene to be edited is the IGF2 gene; the target site of the DNA is the G on intron 3 of the IGF2 gene; the concentration of the crotonic acid is 20 mM; Step 4: Transfect the PE protein expression vector prepared in step 1 and the pegRNA expression vector constructed in step 2 into the gene to be edited with crotonic acid added, so that gene editing occurs at the genomic DNA target site.

2. A method for improving gene editing efficiency by adding crotonic acid according to claim 1, characterized in that: In step 3, the IGF2 gene is derived from bovine fibroblasts.

3. A method for improving gene editing efficiency by adding crotonic acid as claimed in claim 1, characterized in that: In step 2, the pegRNA expression vector is constructed as follows: a pegRNA fragment is obtained by annealing single-stranded DNA, and the pegRNA fragment is connected to the expression vector U6-pegRNA-GG-acceptor to obtain the pegRNA expression vector; The nucleotide sequence of the pegRNA is shown in SEQ ID NO.1.

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