Application of small molecule compound PJ34 in improvement of pilot editing efficiency

Abstract

The invention discloses an application of a small molecule compound PJ34 in improvement of leader editing efficiency, the leader editing efficiency can be enhanced by adding PJ34, the method is simple and convenient to operate, modification of leader editor protein or pegRNA is not needed, and testing of different pegRNA sequences through a large number of pre-experiments is not needed. In addition, PJ34 is easy to synthesize and low in price, and has no obvious toxic effect on cells under the concentration of improving the editing efficiency. Compared with other methods for enhancing the editing efficiency, the method for enhancing the editing efficiency by adding the PJ34 avoids transformation of exogenous DNA or RNA of an editing system, and reduces potential safety risks caused by addition of exogenous genetic materials. In addition, the added PJ34 is good in compatibility with an existing editing system and can be combined with other optimization strategies for use, and therefore the pilot editing efficiency is further improved. In conclusion, a convenient and efficient way is provided for improving the editing efficiency of the pilot editing, and a basis is provided for wide application of the pilot editing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene editing, and particularly relates to application of a small molecule compound PJ34 in improving lead editing efficiency. BACKGROUND

[0002] In recent years, gene editing technology has developed rapidly. At present, various gene editing technologies have been reported, including early gene editing technologies represented by zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), and CRISPR-related editing systems (such as CRISPR / Cas9, base editing and lead editing) developed later. Gene editing technology has important application prospects in many fields, including basic research, clinical medicine and agricultural breeding, and has had a small range of preliminary application.

[0003] However, these technologies still have various obvious defects at present, which limit their large-scale application. For example, ZFN is prone to genome instability and cytotoxicity due to off-target cutting, and has low efficiency in gene knock-in operation, so its application range is limited; TALEN is limited in clinical and practical application due to its large protein size and complex delivery system. The CRISPR / Cas system, as the third generation technology, has rapidly popularized due to its simple design and low cost, but its mechanism of relying on double-strand break may still cause safety hazards such as insertion / deletion mutation, affecting the precision and safety of editing. The base editing technology developed later avoids double-strand break and realizes single-base conversion, but its editing window is narrow, there is bystander editing (i.e. non-specific editing of adjacent bases of the target site), and it cannot complete base transversion or large fragment editing, so its function has obvious limitations.

[0004] In order to develop more precise and widely applicable gene editing tools, David Liu's team reported a new type of gene editing technology—lead editing (Prime Editing) in 2019, which realizes safer and more efficient genome editing through innovative design. Lead editing does not need to rely on DNA double-strand break or exogenous donor template, and can accurately complete 12 kinds of base conversion and transversion, and support the insertion or deletion of target fragments. Its core advantage is high specificity and low off-target effect, which significantly reduces the insertion / deletion mutation caused by double-strand break, and expands the editing flexibility. The editing ability of lead editing covers single-base modification and fragment insertion or deletion, which is expected to provide a safer and more reliable solution for genetic diseases and cancer treatment and agricultural breeding and other fields caused by gene mutation. However, there is still a technical bottleneck that restricts the further widespread application of lead editing, which is that its editing efficiency is generally low, so improving the editing efficiency is a difficult problem that needs to be solved in the field of lead gene editing.

[0005] The existing research improves the editing efficiency of the prime editor through multi-dimensional strategies, but it is accompanied by significant increase in technical complexity and operation cost. For example, the optimization of the core element pegRNA needs to consider length, sequence design and stability: adding a structural RNA motif at the 3' end can significantly enhance its stability and resist exonuclease degradation, thereby improving the efficiency of the prime editor, but its design is more complicated than traditional pegRNA, and additional considerations are needed for the addition of structural motifs, optimization of adapter sequences, and compatibility of 3' end primer binding sites and reverse transcription templates; at the same time, fine regulation of the primer binding site and the length of the reverse transcription template still needs a large number of experimental verification, which further increases the development cycle and research and development cost. In addition, in order to improve the editing success rate, researchers add various functional elements to the editing system, such as PE4 / PE5, which adds the sequence of DNA mismatch repair (MMR) inhibitor protein to the original editing element carrier to improve the editing efficiency, but this strategy continues to increase the size of the editing element carrier on the basis of the already very large editing element carrier, making it even more difficult to deliver the editing element carrier, especially in in vivo applications, which faces the dual challenges of delivery efficiency and safety. SUMMARY

[0006] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0007] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0008] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a method for improving the efficiency of the prime editor, which is simple to operate, that is, by adding a small molecule compound PJ34 to the editing system, thereby improving the efficiency of the prime editor.

[0009] To solve the above technical problems, the present application provides the following technical solutions: the small molecule compound PJ34 is N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetamide, molecular formula C 17 H 17 N3O2, molecular weight 295.34, structural formula as shown in formula I;

[0010]

[0011] The small molecule compound PJ34 with the structural formula as shown in formula I is added to the prime editing system to enhance the editing efficiency of the prime editor.

[0012] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the improvement of the editing efficiency of the prime editing includes the improvement of the base substitution, base insertion and base deletion ability of the genomic sequence.

[0013] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the application method of the small molecule compound PJ34 is to add the compound at the same time in the process of transfecting the editor to the cell.

[0014] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the application concentration of the small molecule compound PJ34 in the prime editing system is 2.5-15 μM.

[0015] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the action time of the small molecule compound PJ34 is 48-72 hours.

[0016] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the prime editor includes PEmax, epegRNA and sgRNA.

[0017] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the cell includes mammalian cells.

[0018] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the mammalian cells include suspension cells and adherent cells.

[0019] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the method of introducing the prime editor includes electroporation transfection method and liposome transfection method.

[0020] As a preferred solution of the application of the small molecule compound PJ34 in improving the efficiency of prime editing, wherein: the small molecule compound PJ34 also includes its derivatives, including PJ34 HCl.

[0021] The application has the following beneficial effects:

[0022] The application provides a small molecule compound capable of improving the efficiency of pilot editing, the small molecule compound is PJ34, and by adding the small molecule compound PJ34 or a derivative thereof, the editing efficiency of pilot editing can be significantly improved, and a convenient, effective and new method for the wide application of pilot editing is provided. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0024] Figure 1 The influence of different compounds in the application embodiment 1 and the comparative example 1 on the efficiency of pilot editing is compared.

[0025] Figure 2 The influence of different concentrations of PJ34 in the application embodiment 2 on the editing efficiency and survival rate of K562-BFP cells is shown in the figure.

[0026] Figure 3 The molecular structure of PJ34 in the application embodiment 3 and the influence of PJ34 (10 μM) on the editing efficiency of different gene sites of HEK293T cells are shown.

[0027] Figure 4 The influence of PJ34 (10 μM) on the editing efficiency of different gene sites of K562 cells in the application embodiment 4 is shown. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned purposes, features and advantages of the application more obvious and easy to understand, the specific implementation of the application will be described in detail in combination with the embodiment of the specification.

[0029] In the following description, many specific details are set forth in order to provide a thorough understanding of the application, but the application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the application, therefore the application is not limited by the specific embodiments disclosed below.

[0030] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the application. In this specification, "in one embodiment" does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0031] The raw materials or chemical reagents used in the present application are commercially available in the art without special instructions.

[0032] Example 1

[0033] This example uses an exogenous reporter gene to verify the application of small molecule compound PJ34 in improving the efficiency of lead editing, specifically:

[0034] This example inserts blue fluorescent protein (BFP) into the AAVS1 site of the genome of K562 cells by CRISPR / Cas9-mediated homologous recombination directed repair (HDR) method, and obtains a cell line stably expressing BFP through puromycin screening and flow cytometry verification. The corresponding epegRNA and sgRNA designed according to the editing site can convert BFP to GFP fluorescence. The plasmid pU6-BFP-epegRNA and BFP-sgRNA lead editing vector are constructed by the Golden Gate method, and the BFP to GFP conversion of the reporter gene cells is carried out using these vectors. The results show that this system can realize the editing of BFP to GFP conversion. In this system, the proportion of GFP positive cells after editing is the embodiment of the efficiency of lead editing, and the application effect of PJ34 is verified by this reporter system.

[0035] K562-BFP cells are electroporated using Lonza 4D-Nucleofector cell nucleus transfection system, and the proportion of GFP positive cells after editing is detected by flow cytometry. The specific steps are as follows:

[0036] (1) One day in advance, the K562-BFP cell line is cultured in RPMI-1640 medium containing 10% FBS to the logarithmic growth phase, and the cell activity is ensured to be greater than 95%. The culture medium in the cell culture dish is aspirated, washed with 1xPBS, and then the cells are digested with trypsin. After the cells are detached into single cells, the digestion is terminated, and the cells are counted by trypan blue staining method.

[0037] (2) Preheat the culture medium, and place the nucleofection solution at room temperature in advance.

[0038] (3) Cell counting: take 1x10 6 g, 10 min, discard the supernatant.

[0039] (4) Prepare plasmid: take a sterile EP tube, and add 1000 ng of pCMV-PEmax plasmid, 325 ng of pU6-BFP-epegRNA plasmid and 112.5 ng of BFP-sgRNA plasmid to the K562-BFP tube, mix well to obtain an editing plasmid mixture, and reserve.

[0040] (5) After centrifugation, all the supernatant was completely sucked off, 100 μL of pre-prepared nucleofection solution was added to resuspend the cell precipitate, and then the cell precipitate was completely transferred to the centrifuge tube containing the plasmid mixture, mixed thoroughly, and then added to the bottom of the electroporation cup. Lightly tap to ensure no bubbles. Place the electroporation cup in the corresponding position in the X module, and use the Lonza 4D-Nucleofector to perform nucleofection on the lead editing reporter gene cells.

[0041] (6) Immediately after electroporation, preheated medium was added to the electroporation cup, mixed, and then the K562-BFP cells containing the transferred plasmid were plated in a 96-well plate, ensuring that the number of cells in each well was 5 x 10 4 cells, and the volume was 180 μL.

[0042] (7) After the diluted compound was thoroughly blown, 20 μL of the diluted small molecule compound PJ34 was added to 180 μL of cells and thoroughly blown to make the working concentration of the compound 5 μM. At the same time, the same concentration of DMSO was set as a blank control, and H2O was added to the 1st and 12th columns of the 96-well plate to reduce evaporation. The plate was placed in a 37°C, 5% CO2 incubator for culture.

[0043] (8) Sample collection and detection: After nucleofection for 48 h, the cells were collected and the expression of BFP and GFP in the cells was detected by flow cytometry. The GFP positive rate was used as the lead editing efficiency.

[0044] Comparative Example 1

[0045] The difference between this comparative example and Example 1 is that the type of compound in the nucleofection step is changed to other popular compounds related to DNA damage and repair in the art and the derivative PJ34-HCl of PJ34. The influence of different compounds on the lead editing efficiency is compared with the results of Example 1 as shown in Figure 1 .

[0046] From Figure 1 it can be seen that the small molecule compound PJ34 and the derivative PJ34-HCl of PJ34 of the present application have significant advantages in improving the lead editing efficiency.

[0047] Example 2

[0048] This example is used to explore the influence of different gradient concentrations (2.5, 5, 10, 15 μM) of the small molecule compound PJ34 on the lead editing efficiency. The results are shown in Figure 2 , and specifically:

[0049] The specific steps are as follows:

[0050] (1) One day in advance, the K562-BFP cell line was cultured in RPMI-1640 medium containing 10% FBS to the logarithmic growth phase, and the cell activity was ensured to be greater than 95%. The medium in the cell culture dish was aspirated, and the cells were counted using the trypan blue staining method.

[0051] (2) Preheat the medium, and place the nucleofection solution at room temperature in advance.

[0052] (3) Cell counting, K562-BFP cells were taken 1×10 6 cells of cell suspension, centrifuged at 100g for 10min, and the supernatant was discarded.

[0053] (4) Preparation of plasmid, sterile EP tube, K562-BFP tube, add 1000ng pCMV-PEmax plasmid, 325ng pU6-BFP-epegRNA plasmid and 112.5ng BFP-sgRNA plasmid, mix well to get editing plasmid mixture, standby.

[0054] (5) After centrifugation, all the supernatant was aspirated, and the nucleofection solution prepared in advance was added. 100μL of nucleofection solution was taken to resuspend the cell pellet, and then all were transferred to the centrifuge tube containing the plasmid mixture. After mixing well, it was added to the bottom of the electroporation cup, and gently tapped to ensure no bubbles. The electroporation cup was placed in the corresponding position in the X module, and the Lonza 4D-Nucleofector was used to nucleofect the lead editing reporter gene cells.

[0055] (6) Immediately after electroporation, preheated medium was added to the electroporation cup, mixed well, and then the K562-BFP cells transfected with plasmids were plated in a 96-well plate, ensuring that the number of cells per well was 5×10 4 cells, and the volume was 180μL.

[0056] (7) After the diluted compound was fully blown, 20μL of the diluted small molecule compound PJ34 was added to 180μL of cells, and the working concentration of the small molecule compound PJ34 was 2.5, 5, 10, 15μM. At the same time, DMSO of the same concentration was set. H2O was added to the 1st and 12th columns of the 96-well plate to reduce evaporation, and the plate was placed in a 37℃, 5% CO2 incubator for culture.

[0057] (8) Sample collection and detection: After nucleofection for 48h, high-throughput flow cytometry was used to detect the expression of BFP and GFP in the lead editing reporter gene cells, and the GFP positive rate was used as the lead editing efficiency.

[0058] In summary, to study the dose-dependent effect mechanism of small molecule compound PJ34 on the efficiency of prime editing, the K562-BFP cell system was used to detect the effect of concentration gradient (2.5-15 μM) of PJ34 on editing efficiency and cell activity. Results Figure 2 As shown in the figure, Figure 2 A is the GFP positive rate, Figure 2 B is the cell survival rate. It can be seen that the GFP positive rate increases with the increase of the concentration of PJ34. When the concentration reaches 10 μM, the effect of improving the efficiency of prime editing is the most significant, and at this time it almost does not affect the cell survival rate. This effect is also consistent in other editing systems.

[0059] Example 3

[0060] This example uses human endogenous gene site editing to verify the application of PJ34 to the efficiency of prime editing. Specifically: In order to more widely verify the small molecule compound PJ34 that improves the editing efficiency of the exogenous reporter gene described above in the efficiency of prime editing, we performed prime editing on multiple endogenous genomic sites. These sites include frequently edited sites in the literature, such as the HEK3 site, the FANCF site, the RNF2 site, etc. Disease-related gene sites such as the HBB site and the EXT1 site were also selected. First, we designed the epegRNA and sgRNA sequence information for these sites according to the above selected target points, and constructed the corresponding vectors. The specific sequence information is shown in Table 1.

[0061] Table 1 epegRNA used in the study

[0062]

[0063]

[0064] Specifically, the steps of prime editing human endogenous gene sites and efficiency detection analysis are as follows:

[0065] 1. Lonza 4D-Nucleofector cell nucleofection system was used to perform electroporation on K562 and HEK293T cells. The specific steps are as follows:

[0066] (1) One day in advance, K562 and HEK293T cell lines were cultured to the logarithmic growth phase in DMEM medium or RPMI-1640 medium containing 10% FBS, respectively, to ensure that the cell activity was greater than 95%. The culture medium in the cell culture dish was aspirated, washed with 1xPBS, and then trypsin was added to digest the cells. After the cells were detached into single cells, the digestion was terminated, and the cells were counted using the trypan blue staining method.

[0067] (2) Preheat the medium, and place the nucleofection solution at room temperature in advance.

[0068] (3) Cell counting, 1 x 10 5 cells of K562 cells and HEK293T cells were taken respectively, centrifuged at 100 g for 10 min, and the supernatant was discarded.

[0069] (4) Preparation of plasmid, sterile EP tube, K562 tube, 100 ng pCMV-PEmax plasmid, 32.5 ng epegRNA plasmid, 11.25 ng sgRNA plasmid were added for standby.

[0070] (5) After centrifugation, all the supernatant was completely sucked off, and the pre-prepared nucleofection solution was added. 20 μL of nucleofection solution was taken to resuspend the cell precipitate, and then it was completely transferred to the centrifuge tube containing the plasmid mixture. After mixing well, it was added to the bottom of the electroporation cup, and it was gently tapped to ensure that no bubbles were generated. The electroporation cup was placed in the corresponding position in the Y module, and the Lonza 4D-Nucleofector was used to nucleofect each cell.

[0071] (6) Immediately after nucleofection, preheated medium was added to the electroporation cup, mixed well, and then the K562-BFP and HEK293T-BFP cells transfected with plasmids were plated in a 96-well plate. At the same time, the small molecule compound PJ34 was added to the cells and mixed well, so that the working concentration of the compound was 10 μM. At the same time, DMSO of the same concentration was set as a blank control, and it was placed in a 37℃, 5% CO2 incubator for culture.

[0072] (7) Sample collection and detection: after 48 h of nucleofection, the cells were collected, the supernatant of HEK293T cells was aspirated with a vacuum pump, and PBS was added twice for washing. 50 μL of trypsin was added, and after 2 min, 200 μL of medium was added to terminate digestion and mix well. Genomic DNA was extracted, amplified, and sent for sequencing.

[0073] 2. Extraction of genomic DNA

[0074] The genomic DNA was extracted by a simplified alkaline lysis method. The cell precipitate was collected, 20-30 μL of alkaline lysis solution was added to the cell precipitate, and then it was incubated at 95℃ for 10 min to achieve rapid lysis of the cells and release of the genomic DNA. After incubation, the sample was immediately placed on ice for "quenching" to terminate the reaction and prevent DNA degradation. Then, 20-30 μL of neutralization solution was added, and it was mixed up and down with a pipette to neutralize the alkaline environment and stabilize the DNA structure. Next, the sample was centrifuged at 10,000 rpm for 1 min to precipitate proteins and other impurities, and the genomic DNA was present in the supernatant. Finally, 2 μL of the supernatant was taken for subsequent PCR analysis.

[0075] 3. Polymerase chain reaction (PCR) for NGS sequencing

[0076] For the completion of the amplification of the editing site in genomic DNA to achieve NGS sequencing. The reaction system is shown in Table 2; the reaction program is shown in Table 3, and the sequencing primer used in the reaction is shown in Table 4.

[0077] Table 2 PCR reaction system

[0078]

[0079] Table 3 PCR reaction program

[0080]

[0081] Table 4 Sequencing primers used in the study

[0082]

[0083]

[0084] 4. NGS sequencing

[0085] (1) PCR primer design principle: primer length 18-22 nt; detection site needs to be located in the 3' end of the forward and reverse primer 10-100 bp interval, the length of the amplicon is preferred 150-300 bp; For polyploid species, prefer to choose a conservative region to design a universal primer, and achieve specific differentiation by sequencing and comparing the polymorphic sites between genomes. The primer sequence is based on Table 4; All primers need to add a fixed adapter at the 5' end: forward primer 5'-ggagtga gtacggtgtgc-3'; Reverse primer 5'-gagttggatgctggatgg-3'.

[0086] (2) First round of PCR amplification: Configure the reaction system according to Table 2, and amplify the target fragment according to the thermal cycling program in Table 3. Take 3-5 μL of the product for 1% agarose gel electrophoresis verification to confirm the generation of specific bands.

[0087] (3) Bar code library construction: Use primers carrying sample-specific barcodes for nested PCR amplification to construct Illumina sequencing libraries. After purification of the amplification product, Illumina HiSeq platform is used for double-end sequencing.

[0088] 5. Editing efficiency analysis software

[0089] Use CRISPResso2 to analyze the proportion of base mutations, insertions or deletions in NGS sequencing data.

[0090] The results are shown in Figure 3 , Figure 4 , Figure 3 A is the molecular structure of PJ34, Figure 3 B~Figure 3 F The effect of PJ34 (10 mM) on the editing efficiency of different gene sites in HEK293T cells, Figure 4 A ~ Figure 4 D The effect of PJ34 (10 mM) on the editing efficiency of different gene sites in K562 cells, according to Figure 3 、 4 It can be seen that, in addition to improving the conversion efficiency of BFP to GFP, PJ34 also has a significant improvement effect in human endogenous gene sites, and the editing types improved by PJ34 to improve the efficiency of prime editing include base substitution, base insertion and base deletion.

[0091] In summary, the application discloses the application of small molecule compound PJ34 in improving the efficiency of prime editing. By adding PJ34 to the original editing system, the editing efficiency of prime editing can be enhanced, which is simple to operate, does not affect the delivery efficiency, does not require modification of the editor protein or pegRNA of prime editing, and does not require pre-experimental testing of different pegRNA sequences. Small molecule compound PJ34 is easy to synthesize, low in price, has no obvious toxic effect on cells at a concentration that improves editing efficiency, and provides a convenient and efficient way to improve the editing efficiency of prime editing. At the same time, compared with other methods for improving editing efficiency, the use of small molecule compound PJ34 avoids complex modification of exogenous DNA and RNA of the editing system, and reduces the safety risk caused by the addition of potential exogenous genetic material. In addition, PJ34 has good compatibility with existing editing systems and methods, and can be used in combination with other optimization strategies to further improve the editing efficiency of prime editing.

[0092] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. Use of a small molecule compound PJ34 in improving the efficiency of prime editing, characterized in that: The small molecule compound PJ34 is N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N- dimethylamino)acetamide, molecular formula C 17 H 17 N3O2, molecular weight 295.34, structural formula as shown in formula I; The small molecule compound PJ34 with structural formula as shown in formula I is added to the prime editing system to achieve the enhancement of editing efficiency of prime editing.

2. The use of small molecule compound PJ34 according to claim 1 for improving the efficiency of prime editing, characterized in that: The enhancement of editing efficiency of the prime editing includes the improvement of base substitution, base insertion and base deletion ability of the genomic sequence.

3. The use of small molecule compound PJ34 according to claim 1 for improving the efficiency of prime editing, characterized in that: The application method of the small molecule compound PJ34 is to add the compound simultaneously in the process of transfecting the editor into the cell.

4. The use of small molecule compound PJ34 according to claim 3 for increasing the efficiency of prime editing, characterized in that: The application concentration of the small molecule compound PJ34 in the prime editing system is 2.5-15 μM.

5. The use of the small molecule compound PJ34 according to claim 4 for increasing the efficiency of prime editing, characterized in that: The action time of the small molecule compound PJ34 is 48-72 hours.

6. The use of the small molecule compound PJ34 according to claim 3 for increasing the efficiency of prime editing, characterized in that: The prime editor includes PEmax, epegRNA and sgRNA.

7. The use of a small molecule compound PJ34 according to claim 3 for improving the efficiency of prime editing, characterized in that: The cell includes a mammalian cell.

8. The use of the small molecule compound PJ34 according to claim 7 for increasing the efficiency of prime editing, characterized in that: The mammalian cell includes a suspension cell and an adherent cell.

9. The use of a small molecule compound PJ34 according to claim 6 for increasing the efficiency of prime editing, characterized in that: The method for introducing the prime editor includes electroporation transfection and liposome transfection.

10. The use of a small molecule compound PJ34 according to claim 1 for increasing the efficiency of prime editing, characterized in that: The small molecule compound PJ34 also includes its derivative, including PJ34 HCl.

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