WO2024078626A1

WO2024078626A1 - Method for improving crispr/cas9-mediated genome knock-in editing efficiency

Info

Publication number: WO2024078626A1
Application number: PCT/CN2023/124585
Authority: WO
Inventors: 郭宜君; 黄菲; 叶露萌; 李竑
Original assignee: 南京金斯瑞生物科技有限公司
Priority date: 2022-10-14
Filing date: 2023-10-13
Publication date: 2024-04-18

Abstract

Provided is a method for enhancing gene knock-in efficiency mediated by a CRISPR gene editing system in cells, comprising placing the cells in contact with a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cyclin 7 (Cdc7) inhibitor during the process of performing a gene knock-in operation on the cells. Further provided is a kit for enhancing the gene knock-in efficiency mediated by the CRISPR gene editing system in cells. By means of the combined use of the DNA-PK inhibitor and the Cdc7 inhibitor, the method and kit provided can both significantly improve gene knock-in efficiency in different cell lines, different insertion sites, and different donor template lengths.

Description

A method to improve the efficiency of CRISPR/Cas9-mediated genome knock-in editing

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application number CN202211263292.9 and filing date October 14, 2022, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to a method for improving the efficiency of CRISPR/Cas9-mediated genome knock-in editing, in particular, a method for improving the efficiency of CRISPR/Cas9-mediated genome knock-in editing using a combination of small molecule protease inhibitors. The present invention also relates to a kit including the above-mentioned small molecule protease inhibitors.

Background technique

The CRISPR/Cas9 system is an RNA-guided genome editing tool that provides researchers with a simple method to quickly modify the genomes of various organisms. With the widespread use of the CRISPR/Cas9 system and the fact that precise gene editing is being or will soon be used clinically for a variety of diseases, the call to improve the efficiency of precise genome editing mediated by this system is growing.

During the use of this system, Cas9, under the guidance of guide RNA (gRNA), locates to the target site of genomic DNA and cuts the targeted gene sequence, forming a DNA double-strand break (DSB), and then repairs the DSB through the intracellular repair mechanism. In mammals, there are two main ways for cells to repair DSB: non-homologous end joining (NHEJ) and homologous recombination repair (HDR). The results of gene editing mainly depend on the choice between these two repair pathways. The NHEJ pathway repairs DSB by directly connecting the ends. This repair does not require large fragments of homologous sequence recognition, but only requires 1-5bp base pairing for repair. It is an error-prone repair with poor accuracy. The HDR pathway can repair DSB by inserting a piece of DNA into the editing site through homologous recombination in the presence of a highly homologous DNA template, thereby achieving the purpose of accurately inserting a DNA sequence into a specific genomic site. Although the precise insertion achieved by HDR is quite advantageous, the choice of repair pathway is biased in the biological context. The repair mechanism of mammalian cells will give priority to NHEJ rather than HDR: NHEJ is active throughout the cell cycle, while HDR is limited to the S/G2 phase; NHEJ is faster than HDR; and NHEJ also inhibits the HDR repair pathway. Therefore, there are more and more studies on whether and how to improve the efficiency of CRISPR/Cas9-mediated precise genome insertion editing by regulating cell repair pathways.

Many teams have conducted in-depth research on the regulation of NHEJ and HDR repair pathways under CRISPR/Cas9-mediated gene editing conditions, and have screened small molecule compounds that regulate NHEJ and HDR repair pathways through a large number of experiments. They have discovered and successfully screened functional small molecules that can improve the efficiency of CRISPR/Cas9-mediated HDR in various cell types. Although there are many types of small molecules discovered through screening, these small molecules have certain cell type characteristics. The enhancement degree described by different authors is also different. Therefore, it is urgent to find a small molecule enhancer with a wider range of applications and the ability to significantly improve the efficiency of genome knock-in editing.

Summary of the invention

In one aspect, the present invention provides a method for enhancing the efficiency of gene knock-in mediated by a CRISPR gene editing system in a cell, comprising contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor during the gene knock-in operation on the cell.

In some embodiments, the CRISPR gene editing system includes a Cas9 protein.

In some embodiments, the CRISPR gene editing system further comprises a guide RNA (gRNA) and a donor template for homologous recombination repair (HDR).

In some embodiments, the donor template is a DNA molecule in single-stranded or double-stranded form.

In some embodiments, the DNA-PK inhibitor is selected from a compound of formula (I), a compound of formula (II), and a combination thereof, wherein X in the compound of formula (I) is hydrogen or deuterium:

Preferably, the compound of formula (I) is:

In some embodiments, the Cdc7 inhibitor is a compound of formula (V) or its hydrochloride salt:

In some embodiments, the compound of formula (I) is contacted with the cell at a concentration of not less than 1 μM; the compound of formula (II) is contacted with the cell at a concentration of not less than 0.5 μM; or the compound of formula (V) is contacted with the cell at a concentration of not less than 10 μM.

In some embodiments, the compound of Formula (I) is contacted with the cell at a concentration of 1-30 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 10-50 μM.

In some embodiments, the compound of Formula (II) is contacted with the cell at a concentration of 0.5-5 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 10-50 μM.

In some embodiments, the compound of Formula (I) is contacted with the cell at a concentration of 5-20 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 20-40 μM.

In some embodiments, the compound of Formula (II) is contacted with the cell at a concentration of 2-4 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 20-40 μM.

In some embodiments, the compound of Formula (I) is contacted with the cell at a concentration of 5 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 30 μM.

In some embodiments, the compound of Formula (II) is contacted with the cell at a concentration of 2 μM, and the compound of Formula (V) is contacted with the cell at a concentration of 30 μM.

In some embodiments, the cell is a HEK293T, Jurkat, SJCRH30, H1975, or A549 cell.

In some embodiments, the cell is a T cell.

In some embodiments, the cell is contacted with the compound of formula (I), the compound of formula (II), and the compound of formula (V) during a gene knock-in procedure.

In some embodiments, the gene knock-in operation is performed by transfection, and the cell is contacted with the compound of formula (V) prior to the transfection.

In some embodiments, the gene knock-in operation is performed by transfection, and the compound of formula (I) and/or the compound of formula (II) is contacted with the cells after the transfection step, and the compound of formula (V) is contacted with the cells both before and after the transfection step.

On the other hand, the present invention provides a kit for enhancing the gene knock-in efficiency mediated by the CRISPR gene editing system in cells, comprising a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor.

In some embodiments, the kit further comprises a Cas9 protein or a nucleic acid molecule encoding the Cas9 protein.

In some embodiments, the kit further comprises a guide RNA (gRNA) and/or a donor template for performing homologous recombination repair (HDR).

Preferably, the compound of formula (I) is:

In some embodiments, the cell is a T cell.

The method provided in this article uses a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor in combination, so that the gene knock-in efficiency of the CRISPR gene editing system can be significantly improved in different cell lines, different insertion sites, and different lengths of donor templates, and is superior to some commercial small molecule enhancers.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the cell viability (A) and knock-in efficiency (B) of small molecule 1 at different concentrations.

FIG2 shows the cell viability (A) and knock-in efficiency (B) of small molecule 2 at different concentrations.

FIG3 shows the cell viability (A) and knock-in efficiency (B) of small molecule 3 at different concentrations.

FIG4 shows the cell viability (A) and knock-in efficiency (B) of small molecule 4 at different concentrations.

FIG5 shows the cell viability (A) and knock-in efficiency (B) of small molecule 5 at different concentrations.

Figure 6 shows different incubation methods for small molecule 5. Method ①: cells were cultured in a medium containing small molecule 5 for 24 hours before electroporation, and immediately after electroporation, the cells were placed in a medium without small molecule 5; Method ②: cells were placed in a medium containing small molecule 5 for 24 hours immediately after electroporation; Method ③: cells were placed in a medium containing small molecule 5 for 24 hours before and after electroporation. Small molecule 5 was not contained in the electroporation buffer.

FIG7 shows the cell viability (A) and knock-in efficiency (B) of small molecule 5 under different incubation modes.

Figure 8 shows the effective concentrations of small molecules and different combinations, where "+" indicates addition and "-" indicates no addition.

FIG. 9 shows the cell viability at 4 days under the action of different molecular combinations.

FIG. 10 shows the knock-in efficiency of cells at 7 days under the action of different molecular combinations.

FIG11 shows the design of the action concentration range of different small molecules and their combinations, where “+” indicates addition and “-” indicates no addition.

FIG. 12 shows the cell knock-in efficiency at 7 days under the concentration range of different small molecules and their combinations.

FIG. 13 shows the knock-in efficiency of cells at 7 days using two types of donor templates, dsDNA and ssDNA, under the action of different small molecule combinations.

Figure 14 shows the validation of cell viability at day 3 using dsDNA and plasmid donor templates in a T cell system.

FIG. 15 shows validation of cell knock-in efficiency at 7 days using dsDNA and plasmid donor templates in a T cell system.

FIG16 shows the restriction enzyme cleavage patterns of different cell lines at different sites 3 days after electroporation.

FIG. 17 shows a comparison of cell knock-in efficiency at 3 days after electroporation of different sites in different cell lines.

Figure 18 shows the comparison of cell knock-in efficiency at 7 days under the action of different small molecule combinations and IDT HDR enhancer.

Figure 19 shows the comparison of cell knock-in efficiency at 3 days under the action of small molecule combination and IDT HDR enhancer.

Detailed ways

Unless otherwise defined, 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 term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

The term "and/or" means any one, any two, any three, any more or all of the listed optional elements.

The term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. When "comprising" or "including" is used, unless otherwise specified, the situation consisting of the stated elements, integers or steps is also covered. For example, when referring to a combination or composition "comprising A and B", it is also intended to cover the combination or composition consisting of A and B.

When referring to specific values, unless otherwise specified or implied by the context, it is generally considered that any value within the range of ±10% of the listed value is referred to at the same time. It will be understood by those skilled in the art that due to reasons such as instrument detection accuracy and errors introduced during operation, the values of the test results, operation time, etc. given may vary within a small range. For example, when it is mentioned that cells are cultured for 24 hours, it should be understood that culturing cells for 24 hours ± 2.4 hours is also feasible. For another example, when it is mentioned that the detected knock-in efficiency is 20%, the actual knock-in efficiency may be 20% ± 2%.

"CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology" is a technology that uses Cas nuclease to edit the target gene DNA under the guidance of RNA. The CRISPR gene editing system used in this technology includes Cas nuclease and guide RNA (single-guide RNA, sgRNA or gRNA), Depending on the situation, double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) as a repair template may also be included. A portion of the sequence of sgRNA can bind to the Cas nuclease, and another portion of the sequence (crRNA) can be complementary to a portion of the sequence of the target gene. With the help of the recognition of sgRNA, the Cas nuclease can form a single-stranded or double-stranded incision at a specific site of the target gene. Cells usually repair DNA broken chains in two ways, namely homologous recombination repair mechanism (homology-directed repair, HDR) and non-homologous end joining repair mechanism (non-homologous end joining, NHEJ). In some applications, CRISPR gene editing technology can be used to knock out genes in cells. In this case, it is usually only necessary to consider destroying the normal coding function of the gene, such as causing frameshift mutations or gene fragment deletions, so that products with normal functions (such as proteins) cannot be produced. Usually, after introducing Cas nucleases (such as Cas9) and sgRNA into cells, cells that do not express the products of the gene to be knocked out can be screened out to achieve the purpose of gene knockout. In other applications, CRISPR gene editing technology can be used to knock in cells, that is, to introduce a target gene into the cell genome so that the cell can produce a product with the target gene. In this case, it can be achieved by providing a donor template with the target gene while introducing Cas nuclease and guide RNA into the cell. The donor template sequence may include the target gene sequence and homologous arm sequences on both sides of the target gene. The purpose of introducing the target gene into the genome is achieved by homologous recombination of the homologous arm sequence and the gap site sequence generated by the Cas nuclease on the genome. In this process, the cutting efficiency of Cas nuclease (such as Cas9), the probability of repairing the double-stranded DNA of the cell genome by homologous recombination, and the probability of recombination with the donor template will affect the final gene knock-in efficiency.

In this article, "Cas9 protein" is the key to the nuclease activity of the CRISPR/Cas9 system. It contains two domains with cutting activity: the HNH domain and the RuvC domain. HNH and RuvC can cut the two strands of DNA to form double-strand breaks. The Cas9 protein can be a recombinant Cas9 protein SpCas9 derived from Streptococcus pyogenes. The Cas9 protein can also be a variant of the Cas9 protein, such as the Cas9 protein variants eSpCas9 (1.0), eSpCas9 (1.1) and SpCas9-HF1 with significantly improved specificity obtained by directed modification of the Cas9 protein.

When referring to sgRNA (or gRNA), the term "target sequence" refers to a nucleotide fragment in the cell genome that is complementary to a portion of the sgRNA sequence (crRNA, about 20 bases). With the help of this portion of the sgRNA that is complementary to the target sequence, proteins such as Cas9 can introduce nucleotide sequence changes in the genome at a relatively certain position to achieve the effect of gene knockout or gene knockin. Accordingly, in this article, "sgRNA targeting a specified sequence" means that the target sequence of the sgRNA is the specified sequence.

The term "donor template" in this article refers to the exogenous DNA template used by the homologous recombination repair (HDR) pathway to mediate gene replacement or insertion after the double-strand break of the cell's DNA. The cleavage site of the Cas9 protein is determined by the sgRNA sequence, and the sequences on both sides of the cleavage site are the homologous arm sequences in the donor template. The target gene sequence that needs to be accurately inserted in the middle of the homologous arm is the sequence of the donor template. The "donor template" can be double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), or it can be a single-stranded oligonucleotide (ssODN), or even a circular DNA (such as a plasmid). The inserted target gene can be any gene sequence that you want to insert into the cell. The target gene sequence may include, for example, genes (nucleotide sequences) encoding defective or missing proteins in the recipient individual or target cell; genes encoding proteins with desired biological or therapeutic effects (for example, antibacterial, antiviral or anti-tumor/anti-cancer functions); genes encoding inhibition or reduction The donor template can be a DNA sequence of any length, such as from a few bp to several thousand bp.

The term "gene knock-in operation" refers to the purposeful introduction of a target gene into a cell by means of technical means such as CRISPR gene editing technology. In some cases, the target gene can be randomly integrated into the cell genome. In other cases, the target gene can be integrated into the cell genome at a relatively determined site (for example, by using the above-mentioned sgRNA with a specific sequence). Typically, the target gene can then be expressed in the cell to generate products such as target proteins, which can be used to study the function of the target gene or for preparing protein products. When the above-mentioned CRISPR gene editing system is used for gene knock-in operation, the gene knock-in operation process may, for example, include preparing cells to be knocked into genes, introducing Cas nucleases, gRNAs and donor templates into cells (for example, by electroporation), continuing to culture cells, and optionally screening positive clones and other steps.

The term "knock-in efficiency or knock-in editing efficiency" refers herein to the ratio of cells including a gene to be knocked-in (target gene) in its genome in a cell population after a gene knock-in operation relative to the total number of cells in the cell population. The detection of gene knock-in efficiency can be carried out in a variety of ways. For example, the gene knock-in efficiency can be determined by detecting an expression product expressing the target gene, such as a fluorescent protein. "Improving knock-in efficiency" refers to improving the gene knock-in operation process so as to obtain a higher knock-in efficiency. In the embodiment of improving knock-in efficiency provided herein, a small molecule enzyme inhibitor is added during the gene knock-in operation process. As described below, by adding a DNA-dependent protein kinase (DNA-PK) inhibitor and/or a cell division cycle protein 7 (Cdc7) inhibitor, the gene knock-in efficiency can be significantly improved.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase and a member of the phosphatidylinositol 3-kinase-related kinase (PI3K) family. It is mainly located in the cell nucleus and can repair DNA double-strand breaks by non-homologous end joining. It plays an important role in maintaining genome stability and V(D)J recombination to produce antibody diversity. Its activity is regulated by DNA double-strand breaks. In addition, DNA-PK and its components are involved in a variety of other physiological processes, including the regulation of chromatin structure, telomere maintenance, transcriptional regulation, and response to replication stress. Common DNA-PK inhibitors include AZD-7648, KU-57788Torin 2, NU5455, VX-984, Samotolisib, etc.

Cyclin 7 (Cdc7) is a serine/threonine kinase that plays an important role in initiating DNA replication. From the late G1 phase to the S phase, Cdc7 forms a complex with Dbf4 (also known as ASK) and phosphorylates Cdc7 substrates to control the transition from G1 phase to S phase. Common Cdc7 inhibitors include XL413, XL413 hydrochloride, CAY10572 (PHA-767491), Cdc7-IN-1, Cdc7-IN-5, Cdc7-IN-17, LY3177833, etc.

The term "cell transfection" is a technique for introducing exogenous genes into eukaryotic cells. Cell transfection pathways can be roughly divided into three categories: chemical mediation, biological mediation, and physical mediation. There are many chemical-mediated methods, such as the classic calcium phosphate co-precipitation method, liposome transfection method, and various cationic substance-mediated techniques. Biological-mediated methods include the more primitive protoplast transfection and the more common various virus-mediated transfection techniques. Physical-mediated methods mainly include electroporation, microinjection, and gene guns. In this article, "electroporation" refers to electroporation, which uses high pulse voltage to destroy cells. The cell membrane potential allows DNA to be introduced through the small holes formed on the membrane for stable/transient transfection. This method is applicable to all types of cells, but the cell lethality is high and the electroporation experimental conditions need to be optimized according to different cell types.

This article provides a better method for improving the efficiency of CRISPR/Cas9-mediated genome knock-in editing. In some embodiments, the specific implementation of the present invention can be divided into the following steps:

(1) Design and synthesize the corresponding sgRNA according to the target cells and target sites; determine the homology arm sequences at both ends of the donor DNA through the sgRNA sequence, add the DNA sequence that needs to be accurately inserted in the middle of the homology arm, which is the sequence of the donor DNA, and design and synthesize the donor DNA template.

(2) Set the test concentration gradients of small molecules 1, 2, and 5 (structural formulas are shown below) respectively, and incubate the cells for a certain period of time before and after transfection. The incubation conditions of small molecules 1 and 2 are: the cells are cultured in the medium containing the small molecules for more than 24 hours after transfection; the incubation conditions of small molecule 5 are: the cells are cultured in the medium containing the small molecules for less than 24 hours before transfection, and the cells are cultured in the medium containing the small molecules for more than 24 hours after transfection.

(3) Cell samples are collected 48-96 hours after transfection. According to the sequence of the donor DNA, the corresponding method is selected, such as NGS, FACS, microplate reader, Agilent 2100 Bioanalyzer System, etc., to detect the knock-in efficiency of the three small molecules at different concentrations, thereby determining the effective concentrations of each of the three small molecules.

(4) Finally, in the CRISPR/Cas9-mediated genome precision editing experiment, the small molecule 1 or small molecule 2 and small molecule 5 with the determined effective concentration are used together to further improve the knock-in efficiency. The incubation conditions are the same as those in the above step (2).

In some embodiments, when performing a gene knock-in operation, the cells are contacted with small molecule 1 and small molecule 5. For example, after transfection (such as electroporation transfection, referred to as electrotransfection), the cells are cultured in a culture medium containing small molecule 1 and small molecule 5 for a period of time; or before transfection, the cells are cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electrotransfection), the cells are cultured in a culture medium containing small molecule 1 and small molecule 5 for a period of time; or before transfection, the cells are cultured in a culture medium containing small molecule 1 and small molecule 5 for a period of time, and after transfection (such as electrotransfection), the cells are cultured in a culture medium containing small molecule 1 and small molecule 5 for a period of time. Preferably, before transfection, the cells are cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electrotransfection), the cells are cultured in a culture medium containing small molecule 1 and small molecule 5 for a period of time.

In some embodiments, when performing a gene knock-in operation, the cell is contacted with small molecule 2 and small molecule 5. For example, after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time; or before transfection, the cell is cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time; or before transfection, the cell is cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time, and after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time. Preferably, the cell is cultured in a culture medium containing small molecule 5 for a period of time before transfection, and the cell is cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time after transfection (such as electroporation).

In some embodiments, when performing a gene knock-in operation, the cell is contacted with small molecule 1, small molecule 2, and small molecule 5. For example, after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time; or before transfection, the cell is cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time; or before transfection, the cell is cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time, and after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time. Preferably, before transfection, the cell is cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electroporation), the cell is cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time.

"Cultivating for a period of time" used herein refers to cultivating a few minutes, a few hours, more than ten hours, or longer time, for example, 30min, 60min, 1h, 2h, 5h, 10h, 20h, 24h, 48h, 72h, and any duration between the listed durations. Preferably, "cultivating for a period of time" refers to cultivating 24-72h, for example 24h.

In some embodiments, when the medium contains small molecule 1, its concentration is not less than 1 μM, for example, so that the concentration of small molecule 1 in the medium is 1-30 μM, such as 5-20 μM. Preferably, the concentration of small molecule 1 in the medium is 5 μM.

In some embodiments, when the medium contains small molecule 2, its concentration is not less than 0.5 μM, for example, so that the concentration of small molecule 2 in the medium is 0.5-5 μM, such as 2-4 μM. Preferably, the concentration of small molecule 2 in the medium is 2 μM.

In some embodiments, when the medium contains small molecule 5, its concentration is not less than 10 μM, for example, so that the concentration of small molecule 5 in the medium is 10-50 μM, such as 20-40 μM. Preferably, the concentration of small molecule 5 in the medium is 30 μM.

Also provided herein is a kit for improving the efficiency of CRISPR/Cas9-mediated genome knock-in editing, including small molecule 1, small molecule 2, small molecule 5, and any combination thereof. In some embodiments, the kit includes small molecule 1 and small molecule 5. In other embodiments, the kit includes small molecule 2 and small molecule 5. In other embodiments, the kit includes small molecule 1, small molecule 2, and small molecule 5. In some embodiments, the kit also optionally includes Cas9 protein or its encoding nucleic acid molecule. In some embodiments, the kit also optionally includes guide RNA (gRNA) and/or a donor template for homologous recombination repair (HDR).

In this paper, by combining DNA-dependent protein kinase (DNA-PK) inhibitors and cell division cycle protein 7 (Cdc7) inhibitors, the gene knock-in efficiency can be better improved in different cell lines, different sites and different donor template lengths, which is better than some commercial small molecule enhancers.

The technical scheme of the present invention is further described in detail below by examples and in conjunction with the accompanying drawings. Unless otherwise specified, the methods and materials of the embodiments described below are conventional products that can be purchased from the market. It will be understood by those skilled in the art that the present invention belongs to that the methods and materials described below are only exemplary and should not be considered as limiting the scope of the present invention.

The implementation of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.

Embodiment 1:

plan:

According to relevant literature, 5 small molecules were selected (the structural formula of the small molecules is shown below) and the concentration ranges of the 5 small molecules were tested respectively. In HEK293T or Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene and flow cytometry was used to verify the knock-in efficiency of the RAB11A site at different concentrations of each small molecule, thereby determining the concentration of each small molecule.

method:

1) Cell culture

Cells were passaged every 2-3 days, and cells required for electroporation were plated 1-2 days before electroporation, and the cell confluence was ensured to reach 70-90% on the day of electroporation.

2) Electroporation

2.1) Add a certain amount of culture medium to the well plate (as shown in Table 1)

Table 1

2.2) Add different amounts of small molecules to the culture medium of the well plate (the final concentration of the small molecules in the culture medium is shown in Table 2, and the structural formula of the small molecules is shown below), and then place the well plate in a 37° C. incubator for preheating.

Table 2

Small molecule structure

Small molecule 1:

Small molecule 2:

Small molecule 3:

Small molecule 4:

Small molecule 5:

2.3) RNP formation: Add Cas9 protein (GenScript, Z03469), RAB11A-sgRNA (SEQ ID NO: 1) (GenScript, EasyEdit), RAB11A-dsDNA template (SEQ ID NO: 2) (GenScript) and buffer R (Thermo Fisher) as shown in Table 3 into a new, RNase-free centrifuge tube and incubate at room temperature for 5-15 min to form RNP.

table 3

2.4) Electroporation system: Add 6 μl of cells resuspended in buffer R (HEK293T: 0.2M; Jurkat: 0.5M) to the above centrifuge tube containing RNPs, and mix by carefully pipetting with a pipette.

2.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters (HEK293T cells: Voltage: 1200 V, Width: 10 ms, Pulse: 3 pulses; Jurkat cells: Voltage: 1600 V, Width: 10 ms, Pulse: 3 pulses).

2.6) Use a 10μl Neon electroporation pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture in the centrifuge tube. Be careful not to have bubbles in the tip. Then insert the Neon electroporation pipette tip into the Neon electroporation tube with 3ml buffer E (Thermo Fisher) added, and click the "Start" button on the Neon electroporation instrument screen.

2.7) Immediately transfer the electroporated samples to the culture medium of the well plate containing different small molecule concentrations. After the electroporation, place the well plate in a 37°C/5% CO2 incubator for further culture.

3) Small molecule mode of action

Within 72 hours after Neon electroporation, the cells were cultured in a medium containing small molecules; after 72 hours, the cells were switched from a medium containing small molecules to a medium without small molecules.

4) Method for detecting knock-in efficiency: Flow cytometry (Beckman) was used to detect the cell viability 3 days after electroporation and the percentage of GFP+ cells (i.e., knock-in efficiency) 7 days after electroporation.

Experimental results:

As shown in Figures 1-5, small molecules 1, 2, and 5 significantly improved the gene knock-in efficiency, and their effective concentrations were: small molecule 1: ≥1μM; small molecule 2: ≥0.5μM; small molecule 5: ≥10μM. Moreover, these three small molecules had no significant effect on cell viability within the effective concentration.

Embodiment 2:

plan:

In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A locus was detected by flow cytometry to optimize the incubation conditions of small molecule 5.

method:

1) Different incubation methods of small molecule 5 are shown in FIG6 .

2) Cell culture

The cells were passaged every 2-3 days, and the cells required for electroporation were plated 1 day before electroporation. When cells were plated before electroporation, two cells were prepared, one of which was cultured without small molecule 5, and the other was cultured with small molecule 5 added to a final concentration of 30 μM 24 hours before electroporation.

3) Electroporation

3.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

3.2) According to the concentration and mode of action of the small molecule in FIG6 , small molecule 5 with a final concentration of 30 μM was added to the culture medium of some wells of the well plate, and then the well plate was placed in a 37° C. incubator for preheating.

3.3) RNP formation: eSpCas9 protein (GenScript, Z03470-300), RAB11A-sgRNA (SEQ ID NO: 1) (GenScript, EasyEdit), RAB11A-dsDNA template (SEQ ID NO: 2) (GenScript) and buffer R (Thermo Fisher) were added to a new, RNase-free centrifuge tube as shown in Table 3 (Jurkat cells) and incubated at room temperature for 5-15 min to form RNP.

3.4) Electroporation system: Add 6 μl of 0.5 M Jurkat cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 uses cells that have not been pre-treated with small molecule 5 in step 2), and the sample group with small molecule 5 uses cells that have been pre-treated with 30 μM small molecule 5 in step 2)), and mix carefully by pipetting with a pipette tip.

3.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 20 ms, Pulse: 1 pulses.

3.6) Use a 10μl Neon electroporation pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture in the centrifuge tube. Be careful not to have bubbles in the tip. Then insert the Neon electroporation pipette tip into the Neon electroporation tube with 3ml buffer E (Thermo Fisher) added, and click the "Start" button on the Neon electroporation instrument screen.

3.7) Immediately transfer the electroporated samples to the corresponding medium wells containing small molecules. After electroporation, place the well plate in a 37°C/5% _CO2 incubator for further culturing for 24 hours, and then transfer the cells to medium without small molecules for culturing.

4) Method for detecting knock-in efficiency: Flow cytometry (Beckman) was used to detect the cell viability 3 days after electroporation and the percentage of GFP+ cells (ie, knock-in efficiency) 6 days after electroporation (as shown in FIG. 7 ).

Experimental results:

As shown in Figure 7, under the premise of no significant effect on cell viability, the optimal incubation condition for small molecule 5 is incubation method ③: cells are cultured in the medium containing small molecule 5 for less than 24 h before electroporation; cells are cultured in the medium containing small molecule 5 after electroporation. The cells were cultured in a medium containing small molecule 5 for 24 h. After 24 h, the cells were switched from a medium containing small molecule 5 to a medium not containing small molecule 5.

Embodiment 3:

plan:

In Jurkat cells, five small molecules were used in combination using green fluorescent protein (GFP) as a reporter gene, and the knock-in efficiency of the RAB11A site was detected by flow cytometry to confirm the optimal small molecule combination.

method:

1) The concentration of small molecules and different combinations (as shown in FIG8 ), where “+” indicates addition and “-” indicates no addition.

2) Small molecule incubation time

Small molecule 1, small molecule 2, small molecule 3, small molecule 4: after electroporation, the cells were cultured in a medium containing small molecules for 24 hours; after 24 hours, the cells were switched from the medium containing small molecules to a medium not containing small molecules.

Small molecule 5: Before electroporation, the cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, the cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, the cells are switched from the medium containing small molecules to a medium without small molecules.

3) Cell culture

The cells were passaged every 2-3 days, and the cells required for electroporation were plated 1 day before electroporation. When cells were plated before electroporation, two aliquots of cells were prepared, one of which was cultured without adding small molecules, and the other aliquot of cells was added with small molecules at a final concentration of 30 μM within 24 hours before electroporation.

4) Electroporation

4.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

4.2) According to the concentration of small molecules and different combinations, different small molecules are added to the culture medium of some wells of the well plate, and then the well plate is placed in a 37° C. incubator for preheating.

4.3) RNP formation: eSpCas9 protein (GenScript, Z03470-300), RAB11A-sgRNA (SEQ ID NO: 1) (GenScript, EasyEdit), RAB11A-dsDNA template (SEQ ID NO: 2) (GenScript) and buffer R (Thermo Fisher) were added to a new, RNase-free centrifuge tube as shown in Table 3 (Jurkat cells) and incubated at room temperature for 5-15 min to form RNP.

4.4) Electroporation system: Add 6 μl of 0.5 M Jurkat cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 used cells not pre-treated with small molecule 5, and the sample group with small molecule 5 used cells pre-treated with 30 μM small molecule 5), and mix by carefully pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 20 ms, Pulse: 1 pulses.

4.6) Use a 10μl Neon electroporation pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture in the centrifuge tube. Be careful not to have bubbles in the tip. Then insert the Neon electroporation pipette tip into the Neon electroporation tube with 3ml buffer E (Thermo Fisher) added, and click the "Start" button on the Neon electroporation instrument screen.

4.7) Immediately transfer the electroporated samples to the corresponding medium wells containing small molecules. After electroporation, place the well plate in a 37°C/5% _CO2 incubator for further 24 hours, and then transfer the cells to medium without small molecules for culturing.

5) Method for detecting knock-in efficiency: Flow cytometry (Beckman) was used to detect the cell viability 4 days after electroporation (as shown in FIG9 ), and the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in FIG10 ).

Experimental results:

As shown in FIG10 , compared with the use of small molecule 1 alone, the knock-in efficiency was increased by 20.2% when small molecule 1 and small molecule 5 were used in combination; compared with the use of small molecule 2 alone, the knock-in efficiency was increased by 22.9% when small molecule 2 and small molecule 5 were used in combination.

The combined use of small molecule 1 and small molecule 5, as well as the combined use of small molecule 2 and small molecule 5, can maximize the knock-in efficiency while ensuring cell viability.

Embodiment 4:

plan:

In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and flow cytometry was used to detect the knock-in efficiency of the RAB11A site, further verifying that the two optimal small molecule combinations in Example 3 had a wider applicable concentration range.

method:

1) The effective concentration range of small molecules is shown in FIG11 , where “+” indicates addition and “-” indicates no addition.

2) Small molecule incubation time

Small molecule 1 and small molecule 2: After electroporation, the cells were cultured in a medium containing small molecules for 24 hours; after 24 hours, the cells were switched from the medium containing small molecules to a medium not containing small molecules.

3) Cell culture

The cells were passaged every 2-3 days, and the cells required for electroporation were plated one day before electroporation. When cells were plated before electroporation, four cells were prepared, one of which was cultured without small molecules, and the other three were added with small molecules at final concentrations of 20μM, 30μM, and 40μM within 24h before electroporation.

4) Electroporation

4.4) Electroporation system: Add 6 μl of 0.5 M Jurkat cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 used cells not pre-treated with small molecule 5, the sample group with 20 μM small molecule 5 used cells pre-treated with 20 μM small molecule 5, the sample group with 30 μM small molecule 5 used cells pre-treated with 30 μM small molecule 5, and the sample group with 40 μM small molecule 5 used cells pre-treated with 40 μM small molecule 5), and mix by carefully pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1600 V, Width: 10 ms, Pulse: 3 pulses.

4.7) Immediately transfer the electroporated samples to the corresponding medium wells containing small molecules. After electroporation, place the well plate in a 37°C/5% CO2 incubator for further culturing for 24 hours, and then transfer the cells to medium without small molecules for culturing.

5) Method for detecting knock-in efficiency: Flow cytometer (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (ie, knock-in efficiency, as shown in FIG. 12 ).

Experimental results

As shown in Figure 12, compared with the use of 5μM small molecule 1 alone, the combined use of 5μM small molecule 1 and small molecule 5 (20-40μM) increased the knock-in efficiency by 31.0%; compared with the use of 20μM small molecule 1 alone, the combined use of 20μM small molecule 1 and small molecule 5 (20-40μM) increased the knock-in efficiency by 21.3%.

Compared with the use of 2μM small molecule 2 alone, the combined use of 2μM small molecule 2 and small molecule 5 (20-40μM) increased the knock-in efficiency by 33.4%; compared with the use of 4μM small molecule 2 alone, the combined use of 4μM small molecule 2 and small molecule 5 (20-40μM) increased the knock-in efficiency by 21.5%.

The combination of small molecule 1 and small molecule 5, and the combination of small molecule 2 and small molecule 5, these two optimal small molecule combinations have a wider applicable concentration range.

Embodiment 5:

plan:

In SJCRH30 cells, green fluorescent protein (GFP) was used as a reporter gene, and flow cytometry was used to detect the knock-in efficiency of the RAB11A site. Two types of templates, dsDNA and ssDNA, were used to further verify that the combination of these two small molecules had a wide range of applicability.

method:

1) Small molecule action plan

① No small molecule; ② 5μM small molecule 1; ③ 2μM small molecule 2; ④ 30μM small molecule 5; ⑤ 5μM small molecule 1 + 30μM small molecule 5; ⑥ 2μM small molecule 2 + 30μM small molecule 5.

2) Small molecule incubation time

3) Cell culture

4) Electroporation

4.1) Add 1 mL of culture medium (RPMI 1640 containing 10% FBS) to a 12-well plate.

4.3) RNP formation: eSpCas9 protein (GenScript, Z03470-300), RAB11A-sgRNA (SEQ ID NO: 1) (GenScript, SafeEdit), RAB11A-dsDNA template or RAB11A-ssDNA template (SEQ ID NO: 2) (GenScript) and buffer R (Thermo Fisher) were added to a new, RNase-free centrifuge tube as shown in Table 4 and incubated at room temperature for 5-15 min to form RNP.

Table 4

4.4) Electroporation system: Add 6 μl of 0.2 M SJCRH30 cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 used cells not pre-treated with small molecule 5, and the sample group with small molecule 5 used cells pre-treated with 30 μM small molecule 5), and mix by carefully pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1200 V, Width: 20 ms, Pulse: 3 pulses.

4.7) Immediately transfer the electroporated samples to the culture medium of the corresponding wells containing small molecules. After electroporation, place the well plate in a 37°C/5% _CO2 incubator for further 24 hours, and then transfer the cells to the culture medium without small molecules for culturing.

5) Method for detecting knock-in efficiency: Flow cytometer (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (ie, knock-in efficiency, as shown in FIG. 13 ).

Experimental results

As shown in Figure 13, for the dsDNA template system, compared with using small molecule 1 or 2 alone, the knock-in efficiency was increased by 9.85% and 10.9% when small molecule 1 or 2 and small molecule 5 were used in combination, respectively; for the ssDNA template system, compared with using small molecule 1 or 2 alone, the knock-in efficiency was increased by 23.2% and 21.4% when small molecule 1 or 2 and small molecule 5 were used in combination, respectively.

At the RAB11A site in SJCRH30 cells, the combined use of small molecule 1 or small molecule 2 and small molecule 5 can increase the knock-in efficiency by 68.7% (dsDNA template) and 46% (ssDNA template) compared to not adding small molecules.

Embodiment 6:

plan:

In T cells, green fluorescent protein (GFP) was used as a reporter gene, and flow cytometry was used to detect the knock-in efficiency of the TRAC site. Two types of templates, dsDNA and plasmid, were used to further verify that the combination of these two small molecules had a wide range of applicability.

method:

1) Small molecule action plan

① No small molecule; ② 5μM small molecule 1 + 30μM small molecule 5; ③ 2μM small molecule 2 + 30μM small molecule 5.

2) Small molecule incubation time

3) Cell isolation, purification, activation and culture

T cells were isolated and purified from 100M PBMC (AllCells) (Thermo Fisher), and the purified T cells were activated using a T cell activation kit (Thermo Fisher). The activated T cells were cultured in TexMACS ^™ Medium (Miltenyi Biotec) containing IL2, IL7, and IL15.

Two cells need to be prepared, one of which is cultured without adding small molecules, and the other is cultured with small molecule 5 added to it at a final concentration of 30 μM within 24 hours before electroporation.

4) Electroporation

4.1) Add 700 μL of culture medium (TexMACS containing 5% FBS, IL2, IL7, IL15) to a 48-well plate.

4.3) RNP formation: Add TrueCut HiFi Cas9 protein (Thermo Fisher), TRAC-sgRNA (SEQ ID NO: 3) (GenScript, SafeEdit), TRAC-dsDNA template (SEQ ID NO: 8) or TRAC plasmid template (SEQ ID NO: 9) (GenScript) and buffer R (Thermo Fisher) as shown in Table 5 into a new, RNase-free centrifuge tube and incubate at room temperature for 5-15 min to form RNP.

table 5

4.4) Electroporation system: Add 6 μl of 0.5M T cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 uses cells not pre-treated with small molecule 5, and the sample group with small molecule 5 uses cells pre-treated with 30 μM small molecule 5), and mix by carefully pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 10 ms, Pulse: 3 pulses.

5) Method for detecting knock-in efficiency: Flow cytometer (Beckman) was used to detect the cell viability 3 days after electroporation (as shown in FIG. 14 ), and the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in FIG. 15 ).

Experimental results

As shown in Figure 15, at the TRAC site in T cells, compared with not adding small molecules, the combined use of small molecule 1 or small molecule 2 and small molecule 5 can increase the knock-in efficiency by 90%-150%, regardless of whether it is a dsDNA or plasmid type template.

Embodiment 7:

plan:

A 6bp HindIII restriction endonuclease recognition sequence was inserted into the HPRT site of Jurkat cells and SJCRH30 cells; a 33bp HiBiT tag was inserted into the VEGFA site of H1975 cells and A549 cells. The method of detecting knock-in efficiency by enzyme digestion, gel running, and Agilent 2100 Bioanalyzer System further verified that the combination of these two small molecules has a wide range of applicability in improving knock-in efficiency.

method:

1) Small molecule action plan

2) Small molecule incubation time

3) Cell culture

Cells were passaged every 2-3 days, and cells required for electroporation were plated one day before electroporation. Two aliquots of each cell type were prepared before electroporation, one of which was cultured without small molecules, and the other was cultured with small molecules 5 added to a final concentration of 30 μM within 24 hours before electroporation.

4) Electroporation

4.1) Add a certain amount of culture medium to the well plate (as shown in Table 6):

Table 6

4.3) RNP formation: eSpCas9 protein (GenScript, Z03470-300), HPRT-sgRNA (SEQ ID NO: 10) or VEGFA-sgRNA (SEQ ID NO: 11) (GenScript, SafeEdit), HPRT-ssDNA template (SEQ ID NO: 12) or VEGFA-ssDNA template (SEQ ID NO: 13) (GenScript) and buffer R (Thermo Fisher) were added to a new, RNase-free centrifuge tube as shown in Table 7 and incubated at room temperature for 5-15 min to form RNP.

Table 7

4.4) Electroporation system: Add 6 μl of cells resuspended in buffer R (Jurkat: 0.5 M; SJCRH30: 0.2 M; H1975: 0.2 M; A549: 0.2 M) to the above centrifuge tube containing RNP (the sample group without small molecule 5 used cells not pre-treated with small molecule 5, and the sample group with small molecule 5 used cells pre-treated with 30 μM small molecule 5), and mix carefully by pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Jurkat: Voltage: 1600V, Width: 10ms, Pulse: 3pulses; SJCRH30: Voltage: 1200V, Width: 20ms, Pulse: 3pulses; H1975: Voltage: 1400V, Width: 20ms, Pulse: 2pulses; A549: Voltage: 1200V, Width: 20ms, Pulse: 3pulses.

5) Methods for detecting knock-in efficiency

5.1) Samples were taken 3 days after electroporation and washed twice with PBS buffer to remove as much PBS buffer as possible from the cells;

5.2) Add 50 μl of cell lysis buffer (Lucigen) to the cells, mix well by pipetting with a pipette tip, and transfer to a PCR tube. The PCR program is as follows:

5.3) Take 4 μl of the above reaction product as a template and perform PCR amplification in a 50 μl system. The PCR system based on KOD FX Polymerase (TOYOBO) is as follows:

HPRT-F/R and VEGFA-F/R were synthesized at Nanjing GenScript Biotechnology Co., Ltd.

PCR procedure:

5.4) The above PCR products were recovered by gel (Tian Gen)

5.5) The concentration of the gel-recovered product was determined using Qubit 1X dsDNA BR (Broad Range) Assay Kits. 100 ng of the gel-recovered product was subjected to enzyme digestion (Jurkat and SJCRH30: HindIII (NEB); H1975 and A549: BsrBI (NEB)). The enzyme digestion system was as follows:

The enzyme digestion procedure is as follows:

5.6) Take 4 μl of the above digestion product and run it on 1% agarose gel. The gel map is shown in Figure 16. The digestion fragments 1 and 2 are consistent with the theoretical values. Take another 1 μl of the above digestion product and use the Agilent 2100 Bioanalyzer System to detect the concentration of each length fragment in the digestion product. The calculation formula for the knock-in efficiency is: (Conc. digestion fragment 1 + Conc. digestion fragment 2) / (Conc. digestion fragment 1 + Conc. digestion fragment 2 + Conc. full length) (as shown in Figure 17).

Experimental results

As shown in Figure 17, at the HPRT site in Jurkat cells, compared with not adding small molecules, the combined use of small molecules can increase the knock-in efficiency by 461% (small molecule 1 + small molecule 5) and 449% (small molecule 2 and small molecule 5); at the HPRT site in SJCRH30 cells, compared with not adding small molecules, the combined use of small molecules can increase the knock-in efficiency by 194% (small molecule 1 + small molecule 5) and 184% (small molecule 2 and small molecule 5); at the VEGFA site in H1975 cells, compared with not adding small molecules, the combined use of small molecules can increase the knock-in efficiency by 82.6% (small molecule 1 + small molecule 5) and 43.6% (small molecule 2 and small molecule 5); at the VEGFA site in A549 cells, compared with not adding small molecules, the combined use of small molecules can increase the knock-in efficiency by 65.3% (small molecule 1 + small molecule 5) and 63.0% (small molecule 2 and small molecule 5).

In general, the two small molecules can greatly improve the efficiency of gene knock-in in different cell lines and different sites, and have wide applicability.

Embodiment 8:

plan:

In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A locus was detected by flow cytometry. The optimized knock-in efficiency was improved by several small molecule solutions and commercial HDR EnhancerV1(IDT) for comparison.

method:

1) Small molecule action plan

Several optimized small molecule solutions with higher knock-in efficiency are as follows: ①5μM small molecule 1; ②2μM small molecule 2; ③5μM small molecule 1+30μM small molecule 5; ④2μM small molecule 2+30μM small molecule 5.

Commercialization of IDT HDR enhancer V1: 30μM (recommended concentration on IDT official website).

2) Small molecule incubation time

Small molecule 1, small molecule 2, IDT HDR enhancer: After electroporation, the cells were cultured in a medium containing small molecules for 24 hours. After 24 hours, the cells were switched from a medium containing small molecules to a medium without small molecules.

3) Cell culture

4) Electroporation

4.7) Immediately transfer the electroporated samples to the culture medium of the corresponding wells. After electroporation, place the well plate in a 37°C/5% CO2 incubator for further culturing for 24 hours, and then transfer the cells to a culture medium without small molecules for culturing.

5) Method for detecting knock-in efficiency: Flow cytometer (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (ie, knock-in efficiency, as shown in FIG. 18 ).

Experimental results

As shown in Figure 18, compared with the use of small molecule 1 or small molecule 2 alone, the knock-in efficiency was increased by about 30% when small molecule 1 or 2 and small molecule 5 were used in combination; compared with IDT HDR enhancer, the knock-in efficiency was increased by about 50% when small molecule 1 or 2 and small molecule 5 were used in combination.

Overall, the optimized small molecule combination performed better than the commercial IDT HDR enhancer in improving gene knock-in efficiency.

Embodiment 9:

plan:

A 6 bp HindIII restriction endonuclease recognition sequence was inserted into the TRAC site of HEK293T cells, and the knock-in efficiency was detected using the Agilent 2100 Bioanalyzer System. The small molecule combination (small molecule 1 + small molecule 5) was further compared with the commercial IDT The role of HDR enhancer V1 in improving knock-in efficiency.

method:

1) Small molecule action plan

The small molecule combination is: 5 μM small molecule 1 + 30 μM small molecule 5;

IDT The concentration of HDR enhancerV1 is: 30μM (recommended concentration on IDT official website).

2) Small molecule incubation time

Small molecules 1. IDT HDR enhancer V1: After electroporation, cells are cultured in a medium containing small molecules for 24 hours; after 24 hours, the cells are switched from a medium containing small molecules to a medium without small molecules.

3) Cell culture

4) Electroporation

4.1) Add 1 mL of culture medium (DMEM containing 10% FBS) to a 12-well plate.

4.3) RNP formation: Add Cas9 protein (Thermo Fisher, A36499), TRAC-sgRNA (SEQ ID NO: 3) (GenScript, EasyEdit), TRAC-86bp dsODN template (annealing product of SEQ ID NO: 4 and 5) (GenScript) and buffer R (Thermo Fisher) as shown in Table 8 into a new, RNase-free centrifuge tube and incubate at room temperature for 5-15 min to form RNP.

Table 8

4.4) Electroporation system: Add 6 μl of 0.2 M HEK293T cells resuspended in buffer R to the above centrifuge tube containing RNP (the sample group without small molecule 5 used cells not pre-treated with small molecule 5, and the sample group with small molecule 5 used cells pre-treated with 30 μM small molecule 5), and mix by carefully pipetting with a pipette tip.

4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1200 V, Width: 10 ms, Pulse: 3 pulses.

5) Methods for detecting knock-in efficiency

5.1) Take samples 3 days after electroporation and wash them twice with PBS buffer to remove as much PBS buffer as possible.

5.2) Add 50 μl of cell lysis buffer (Lucigen) to the cells, mix well by pipetting with a pipette, and transfer to a PCR tube. The PCR program is as follows:

TRAC-F and TRAC-R were synthesized at Nanjing GenScript Biotechnology Co., Ltd.

PCR procedure:

5.4) The above PCR products were recovered by gel (Tian Gen)

5.5) The gel recovery product was measured for concentration using Nanodrop, and 200 ng of the gel recovery product was subjected to HindIII (NEB) digestion. The digestion system was as follows:

The enzyme digestion procedure is as follows:

5.6) Take 1 μl of the above digestion product and use Agilent 2100 Bioanalyzer System to detect the concentration of each length fragment in the digestion product. The calculation formula for the knock-in efficiency is: (Conc. digestion fragment 1 + Conc. digestion fragment 2) / (Conc. digestion fragment 1 + Conc. digestion fragment 2 + Conc. full length) (as shown in Figure 19).

Experimental results

As shown in Figure 19, compared with the IDT HDR enhancer, the knock-in efficiency was increased by 31.9% when small molecules 1 and 5 were used in combination. At the TRAC site of HEK293T cells, the optimized small molecule combination was superior to the commercial IDT HDR enhancer in improving the knock-in efficiency.

The CRISPR/Cas9 system is a widely used genome editing tool that can achieve precise gene editing at specific sites. There are many types of small molecules, but these small molecules have certain cell type specificity and context dependence, and the degree of improvement described by each author also varies. The present invention uses two small molecule compounds together to provide a method with a wide range of applications that can significantly improve the efficiency of CRISPR/Cas9-mediated precision gene editing.

The present invention uses two small molecule compounds in combination to provide a method for better improving the efficiency of CRISPR/Cas9-mediated precise gene editing, the method comprising:

Targeting different target sites in different cells, the knock-in efficiency of small molecules 1, 2, and 5 at different concentrations was verified to determine the optimal concentration of each small molecule under the conditions of the cell line and site.

The small molecule 1 or small molecule 2 and small molecule 5 at the optimal concentration are used in combination to improve the knock-in efficiency at different sites in the above-mentioned different cells.

The present invention uses small molecule 1 or small molecule 2 and small molecule 5 in combination, which can better improve the knock-in efficiency in different cell lines, different sites and different donor lengths, and is superior to some commercial small molecule enhancers.

The nucleotide sequences mentioned in this article are as follows:

Claims

A method for enhancing the efficiency of gene knock-in mediated by a CRISPR gene editing system in a cell, comprising contacting the cell with a DNA-dependent protein kinase inhibitor and a cell division cycle protein 7 inhibitor during the gene knock-in operation on the cell.
The method of claim 1, wherein the CRISPR gene editing system comprises a Cas9 protein.
The method of claim 1 or 2, wherein the CRISPR gene editing system further comprises a guide RNA and a donor template for homologous recombination repair.
The method according to any one of claims 1 to 3, wherein the donor template is a DNA molecule in single-stranded or double-stranded form.
The method according to any one of claims 1 to 4, wherein the DNA-dependent protein kinase inhibitor is selected from a compound of formula (I), a compound of formula (II), and a combination thereof, wherein X in the compound of formula (I) is hydrogen or deuterium:

Preferably, the compound of formula (I) is:
The method according to any one of claims 1 to 5, wherein the cell division cycle protein 7 inhibitor is a compound of formula (V) or its hydrochloride:
The method of claim 5 or 6, wherein the compound of formula (I) is contacted with the cells at a concentration of not less than 1 μM; the compound of formula (II) is contacted with the cells at a concentration of not less than 0.5 μM; or the compound of formula (V) is contacted with the cells at a concentration of not less than 10 μM.
The method according to any one of claims 5 to 7, wherein:

1) the compound of formula (I) is contacted with the cells at a concentration of 1-30 μM, and the compound of formula (V) is contacted with the cells at a concentration of 10-50 μM; or

2) The compound of formula (II) is contacted with the cells at a concentration of 0.5-5 μM, and the compound of formula (V) is contacted with the cells at a concentration of 10-50 μM.
The method according to any one of claims 5 to 8, wherein:

1) the compound of formula (I) is contacted with the cells at a concentration of 5-20 μM, and the compound of formula (V) is contacted with the cells at a concentration of 20-40 μM; or

2) The compound of formula (II) is contacted with the cells at a concentration of 2-4 μM, and the compound of formula (V) is contacted with the cells at a concentration of 20-40 μM.
The method according to any one of claims 5 to 9, wherein:

1) the compound of formula (I) is contacted with the cells at a concentration of 5 μM, and the compound of formula (V) is contacted with the cells at a concentration of 30 μM; or

2) The compound of formula (II) is contacted with the cells at a concentration of 2 μM, and the compound of formula (V) is contacted with the cells at a concentration of 30 μM.
The method of any one of claims 1 to 10, wherein the cell is a HEK293T, Jurkat, SJCRH30, H1975 or A549 cell.
The method of any one of claims 1 to 11, wherein the cell is a T cell.
The method according to any one of claims 5 to 12, wherein the cell is contacted with the compound of formula (I), the compound of formula (II) and the compound of formula (V) during the gene knock-in operation on the cell.
The method according to any one of claims 5 to 13, wherein the gene knock-in operation is performed by transfection, and the cells are contacted with the compound of formula (V) before the transfection.
The method according to any one of claims 5 to 14, wherein the gene knock-in operation is performed by transfection, the compound of formula (I) and/or the compound of formula (II) is contacted with the cells after the transfection step, and the compound of formula (V) is contacted with the cells both before and after the transfection step.
A kit for enhancing the efficiency of gene knock-in mediated by the CRISPR gene editing system in cells, including a DNA-dependent protein kinase inhibitor and a cell division cycle protein 7 inhibitor.
The kit as claimed in claim 16, wherein the kit further comprises a Cas9 protein or a nucleic acid molecule encoding it.
The kit of claim 16 or 17, wherein the kit further comprises a guide RNA and/or a donor template for homologous recombination repair.
The kit according to any one of claims 16 to 18, wherein the donor template is a DNA molecule in single-stranded or double-stranded form.
The kit according to any one of claims 16 to 19, wherein the DNA-dependent protein kinase inhibitor is selected from a compound of formula (I), a compound of formula (II), and a combination thereof, wherein X in the compound of formula (I) is hydrogen or deuterium:

Preferably, the compound of formula (I) is:
The kit according to any one of claims 16 to 20, wherein the cell division cycle protein 7 inhibitor is a compound of formula (V) or its hydrochloride:
The kit according to any one of claims 16 to 21, wherein the cell is a HEK293T, Jurkat, SJCRH30, H1975 or A549 cell.
The kit of any one of claims 16 to 22, wherein the cell is a T cell.