Method for improving CRISPR / Cas9 mediated genome knock-in editing efficiency

Abstract

The invention provides a method for enhancing gene knock-in efficiency mediated by a CRISPR gene editing system in a cell. The method comprises the step of enabling the cell to be in contact with a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor in the gene knock-in operation process of the cell. The invention also provides a kit for enhancing the gene knock-in efficiency mediated by the CRISPR gene editing system in cells. According to the method and the kit provided by the invention, the DNA-PK inhibitor and the Cdc7 inhibitor are combined for use, so that the gene knock-in efficiency can be remarkably improved under the conditions of 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

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number CN202211263292.9, filed on October 14, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] This article relates to methods for improving the efficiency of CRISPR / Cas9-mediated genome knock-in editing, particularly methods for improving the efficiency of CRISPR / Cas9-mediated genome knock-in editing using a combination of small molecule protease inhibitors. This article also relates to kits comprising the above-mentioned small molecule protease inhibitors. Background Art

[0004] The CRISPR / Cas9 system is an RNA-guided genome editing tool that provides researchers with a simple and rapid method for modifying the genomes of various organisms. With the widespread use of the CRISPR / Cas9 system and its precise gene editing being or soon to be used clinically for a variety of diseases, the call for improving the efficiency of this system-mediated precision genome editing is growing.

[0005] During the use of this system, Cas9, guided by a guide RNA (gRNA), locates the target site in genomic DNA and cuts the targeted gene sequence, forming a DNA double-strand break (DSB). The DSB is then repaired by intracellular repair mechanisms. In mammals, cells repair DSBs through two main pathways: non-homologous end joining (NHEJ) and homologous recombination repair (HDR). The outcome of gene editing depends primarily on the choice between these two repair pathways. The NHEJ pathway repairs DSBs by direct end-to-end ligation. 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 precision. The HDR pathway can repair DSBs by inserting a DNA segment into the editing site through homologous recombination in the presence of a highly homologous DNA template, thereby achieving the goal of precisely inserting a DNA sequence into a specific genomic site. Although the precise insertion achieved by HDR is advantageous, the choice of repair pathway can be biased in biological contexts. Mammalian cells' repair mechanisms prioritize NHEJ over HDR: NHEJ is active throughout the cell cycle, while HDR is restricted to the S / G2 phase; NHEJ is faster than HDR; and NHEJ also inhibits the HDR repair pathway. Consequently, research is increasingly focused on whether and how to improve the efficiency of CRISPR / Cas9-mediated precise genome editing by regulating cellular repair pathways.

[0006] Many teams have conducted in-depth research on the regulation of NHEJ and HDR repair pathways under CRISPR / Cas9-mediated gene editing conditions. Through extensive experimental screening of small molecule compounds that regulate NHEJ and HDR repair pathways, they have discovered and successfully screened for functional small molecules that can enhance the efficiency of CRISPR / Cas9-mediated HDR in various cell types. Although a wide range of small molecules has been discovered through screening, these small molecules have certain cell type specificity and context dependence, and the degree of improvement described by different authors also varies. Therefore, there is an urgent need to find a small molecule enhancer with a wider range of applications that can significantly improve the efficiency of genome knock-in editing.

[0007] Summary of the Invention

[0008] In one aspect, the present invention provides a method for enhancing the efficiency of gene knock-in mediated by the 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.

[0009] In some embodiments, the CRISPR gene editing system includes a Cas9 protein.

[0010] In some embodiments, the CRISPR gene editing system further comprises a guide RNA (gRNA) and a donor template for homologous recombination repair (HDR).

[0011] In some embodiments, the donor template is a DNA molecule in single-stranded or double-stranded form.

[0012] 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:

[0013] Preferably, the compound of formula (I) is:

[0014] In some embodiments, the Cdc7 inhibitor is a compound of formula (V) or its hydrochloride salt:

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments, 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.

[0022] In some embodiments, the cell is a HEK293T, Jurkat, SJCRH30, H1975, or A549 cell.

[0023] In some embodiments, the cell is a T cell.

[0024] 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.

[0025] In some embodiments, the gene knock-in procedure is performed by transfection, and the cells are contacted with the compound of formula (V) prior to the transfection.

[0026] 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) are 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.

[0027] 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.

[0028] In some embodiments, the kit further comprises a Cas9 protein or a nucleic acid molecule encoding the Cas9 protein.

[0029] In some embodiments, the kit further comprises a guide RNA (gRNA) and / or a donor template for performing homologous recombination repair (HDR).

[0030] In some embodiments, the donor template is a DNA molecule in single-stranded or double-stranded form.

[0031] 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:

[0032] Preferably, the compound of formula (I) is:

[0033] In some embodiments, the Cdc7 inhibitor is a compound of formula (V) or its hydrochloride salt:

[0034] In some embodiments, the cell is a HEK293T, Jurkat, SJCRH30, H1975, or A549 cell.

[0035] In some embodiments, the cell is a T cell.

[0036] The method provided in this article significantly improves the gene knock-in efficiency of the CRISPR gene editing system in different cell lines, different insertion sites, and donor templates of different lengths by combining a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cyclin 7 (Cdc7) inhibitor, and is superior to some commercial small molecule enhancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the cell viability (A) and knock-in efficiency (B) of small molecule 1 at different concentrations.

[0038] Figure 2 shows the cell viability (A) and knock-in efficiency (B) of small molecule 2 at different concentrations.

[0039] FIG3 shows the cell viability (A) and knock-in efficiency (B) of small molecule 3 at different concentrations.

[0040] FIG4 shows the cell viability (A) and knock-in efficiency (B) of small molecule 4 at different concentrations.

[0041] FIG5 shows the cell viability (A) and knock-in efficiency (B) of small molecule 5 at different concentrations.

[0042] Figure 6 shows different incubation methods for small molecule 5. Method 1: cells were cultured in medium supplemented with small molecule 5 for 24 hours before electroporation, and immediately after electroporation, the cells were incubated in medium without small molecule 5. Method 2: cells were immediately incubated in medium supplemented with small molecule 5 for 24 hours after electroporation. Method 3: cells were incubated in medium supplemented with small molecule 5 for 24 hours before and after electroporation. Small molecule 5 was not included in the electroporation buffer.

[0043] FIG7 shows the cell viability (A) and knock-in efficiency (B) of small molecule 5 under different incubation modes.

[0044] Figure 8 shows the effective concentrations of small molecules and different combinations, where "+" indicates addition and "-" indicates no addition.

[0045] FIG9 shows the cell viability after 4 days under the action of different molecular combinations.

[0046] FIG10 shows the knock-in efficiency of cells under the action of different molecular combinations at 7 days.

[0047] Figure 11 shows the design of the effective concentration ranges of different small molecules and their combinations, where "+" indicates addition and "-" indicates no addition.

[0048] FIG12 shows the cell knock-in efficiency at 7 days under the action concentration range of different small molecules and their combinations.

[0049] FIG13 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.

[0050] Figure 14 shows the cell viability at day 3 validation using dsDNA and plasmid donor templates in a T cell system.

[0051] Figure 15 shows the validation of cell knock-in efficiency at 7 days using dsDNA and plasmid donor templates in a T cell system.

[0052] FIG16 shows the enzyme digestion patterns of different cell lines at different sites 3 days after electroporation.

[0053] FIG17 shows a comparison of cell knock-in efficiency 3 days after electroporation of different cell lines at different sites.

[0054] Figure 18 shows the comparison of cell knock-in efficiency after 7 days under the action of different small molecule combinations and IDT HDR enhancer.

[0055] Figure 19 shows the comparison of cell knock-in efficiency after 3 days of treatment with small molecule combination and IDT HDR enhancer. DETAILED DESCRIPTION

[0056] 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.

[0057] The term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

[0058] The term "and / or" means any one, any two, any three, any more or all of the listed optional elements.

[0059] The terms "comprising" or "including" refer to including the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. When "comprising" or "including" is used, unless otherwise indicated, it also covers the situation where it consists of the stated elements, integers, or steps. For example, when it is mentioned that a combination or composition "comprising A and B", it is also intended to cover the combination or composition consisting of A and B.

[0060] When referring to specific numerical values, unless the context otherwise indicates or implies, it is generally considered that any numerical value within the range of ±10% of the listed numerical value is mentioned 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 example, when it is mentioned that the knock-in efficiency detected is 20%, the actual knock-in efficiency may be 20% ± 2%.

[0061] "Clustered Regularly Interspaced Short Palindromic Repeats" gene editing technology is an RNA-guided technique that uses Cas nucleases to edit the DNA of target genes. The CRISPR gene editing system used in this technology includes Cas nucleases and guide RNA (single-guide RNA, sgRNA or gRNA), and may also include double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) as a repair template. Part of the sgRNA sequence can bind to the Cas nuclease, and another part of the sequence (crRNA) can be complementary to part of the target gene sequence. With the help of the recognition effect of the sgRNA, the Cas nuclease can form a single-stranded or double-stranded cut at a specific site in the target gene. Cells typically repair DNA breaks through two methods: homologous recombination repair (HDR) and 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. Generally, after introducing Cas nucleases (such as Cas9) and sgRNA into cells, cells that do not express the product 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 perform gene knock-in operations (knock in) on cells, that is, to introduce the target gene into the cell genome so that the cell can generate products with the target gene. In this case, it can be achieved by providing a donor template with the target gene while introducing Cas nucleases and guide RNA into the cell. The donor template sequence can include the target gene sequence and homology arm sequences on both sides of the target gene. By means of homologous recombination of the homology arm sequence and the gap site sequence generated by the Cas nuclease on the genome, the purpose of introducing the target gene into the genome is achieved. In this process, the cutting efficiency of Cas nuclease (such as Cas9), the probability of repairing double-stranded DNA breaks in the cell genome by homologous recombination, and the probability of recombination with the donor template will all affect the final gene knock-in efficiency.

[0062] The term "Cas9 protein" as used herein is crucial for the nuclease activity of the CRISPR / Cas9 system. It contains two domains with cleavage activity: the HNH domain and the RuvC domain. HNH and RuvC, respectively, cleave the two strands of DNA, forming double-strand breaks. The Cas9 protein can be SpCas9, a recombinant Cas9 protein derived from Streptococcus pyogenes. The Cas9 protein can also be a variant of the Cas9 protein, such as eSpCas9 (1.0), eSpCas9 (1.1), and SpCas9-HF1, Cas9 variants with significantly improved specificity, derived from targeted engineering of the Cas9 protein.

[0063] When referring to sgRNA (or gRNA), the term "target sequence" refers to a nucleotide fragment in the cellular genome that is complementary to a portion of the sgRNA sequence (crRNA, approximately 20 bases). This complementary portion of the sgRNA allows proteins like Cas9 to introduce nucleotide sequence changes at relatively specific locations in the genome, achieving gene knockout or knock-in effects. Accordingly, in this article, "sgRNA targeting a specific sequence" means that the sgRNA's target sequence is that specific sequence.

[0064] The term "donor template" in this article is an exogenous DNA template mediated by the homologous recombination repair (HDR) pathway after double-strand breaks in cell 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 homology arm sequences in the donor template. The target gene sequence that needs to be accurately inserted is added in the middle of the homology arm, which is the sequence of the donor template. "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 is desired to be inserted into the cell. The target gene sequence may include, for example, a gene (nucleotide sequence) encoding a protein that is defective or lacking in a recipient individual or target cell; a gene encoding a protein with a desired biological or therapeutic effect (e.g., antibacterial, antiviral or antitumor / anticancer function); a nucleotide sequence encoding an RNA that suppresses or reduces the production of harmful or otherwise undesirable proteins (e.g., a nucleotide sequence encoding an RNA interfering agent); and / or a nucleotide sequence encoding an antigenic protein. The donor template can be a DNA sequence of any length, such as from a few bp to several thousand bp.

[0065] The term "gene knock-in operation" refers to the purposeful introduction of a target gene into a cell by technical means such as CRISPR gene editing technology in this article. 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 adopting 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 a protein product. When the above-mentioned CRISPR gene editing system is used to perform the gene knock-in operation, the gene knock-in operation process can, 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 steps such as positive clones.

[0066] The term "knock-in efficiency or knock-in editing efficiency" refers herein to the ratio of cells including a gene to be knocked into (target gene) in its genome relative to the total cell number of the cell colony after the gene knock-in operation. 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 the expression product of the target gene, such as fluorescent protein. "Improving knock-in efficiency" refers to improving the gene knock-in operation process to obtain higher knock-in efficiency. In the embodiment of the improvement knock-in efficiency provided herein, small molecule enzyme inhibitors are added during the gene knock-in operation. As described below, by adding DNA-dependent protein kinase (DNA-PK) inhibitors and / or cell division cycle protein 7 (Cdc7) inhibitors, the gene knock-in efficiency can be significantly improved.

[0067] 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 primarily localized in the nucleus and repairs DNA double-strand breaks through non-homologous end joining. It plays a crucial role in maintaining genomic stability and V(D)J recombination, which generates antibody diversity. Its activity is regulated by DNA double-strand breaks. Furthermore, DNA-PK and its components participate in a variety of other physiological processes, including regulation of chromatin structure, telomere maintenance, transcriptional regulation, and responses to replication stress. Common DNA-PK inhibitors include AZD-7648, KU-57788, Torin 2, NU5455, VX-984, and Samotolisib.

[0068] Cyclin 7 (Cdc7) is a serine / threonine kinase that plays an important role in initiating DNA replication. From late G1 phase to S phase, Cdc7 forms a complex with Dbf4 (also known as ASK) and phosphorylates Cdc7 substrates to control the transition from G1 to S phase. Common Cdc7 inhibitors include XL413, XL413 hydrochloride, CAY10572 (PHA-767491), Cdc7-IN-1, Cdc7-IN-5, Cdc7-IN-17, and LY3177833.

[0069] 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 technologies. Biological-mediated methods include the more primitive protoplast transfection and the various virus-mediated transfection techniques that are more common nowadays. Physical mediation methods mainly include electroporation, microinjection, and gene guns. In this article, "electroporation" refers to electroporation, which uses high pulse voltage to destroy the cell membrane potential, allowing DNA to be introduced through small pores formed on the membrane for stable / transient transfection. This method is suitable for all types of cells, but the cell lethality is high, and the electroporation experimental conditions need to be optimized according to different cell types.

[0070] This article provides a 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:

[0071] (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 precisely inserted in the middle of the homology arm, which is the sequence of the donor DNA, and design and synthesize the donor DNA template.

[0072] (2) Set up a test concentration gradient for three small molecules, small molecule 1, small molecule 2, and small molecule 5 (structural formula see below), and incubate the cells for a certain period of time before and after transfection. The incubation conditions for small molecules 1 and 2 are: cells are cultured in medium containing the small molecules for more than 24 hours after transfection; the incubation conditions for small molecule 5 are: cells are cultured in medium containing the small molecule for less than 24 hours before transfection, and cells are cultured in medium containing the small molecule for more than 24 hours after transfection.

[0073] (3) Cell samples are collected 48-96 hours after transfection. Depending on the sequence of the donor DNA, appropriate methods are 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.

[0074] (4) Finally, in the CRISPR / Cas9-mediated precise genome editing experiment, the determined concentrations of small molecule 1 or small molecule 2 and small molecule 5 are used in combination to further improve the knock-in efficiency. The incubation conditions are the same as those in step (2).

[0075] 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.

[0076] In some embodiments, when performing a gene knock-in operation, the cells are contacted with small molecule 2 and small molecule 5. For example, after transfection (such as electroporation), the cells are cultured in a culture medium containing small molecule 2 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 electroporation), the cells are cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time; or before transfection, the cells are 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 cells are cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time. Preferably, the cells are cultured in a culture medium containing small molecule 5 for a period of time before transfection, and after transfection (such as electroporation), the cells are cultured in a culture medium containing small molecule 2 and small molecule 5 for a period of time.

[0077] In some embodiments, when performing a gene knock-in operation, the cells are contacted with small molecule 1, small molecule 2, and small molecule 5. For example, after transfection (such as electroporation), the cells are 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 cells are cultured in a culture medium containing small molecule 5 for a period of time, and after transfection (such as electroporation), the cells are 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 cells are 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 cells are cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time. Preferably, the cells are cultured in a culture medium containing small molecule 5 for a period of time before transfection, and after transfection (such as electroporation), the cells are cultured in a culture medium containing small molecule 1, small molecule 2, and small molecule 5 for a period of time.

[0078] "Cultivating for a period of time" as used herein refers to cultivating for a few minutes, a few hours, more than ten hours, or longer periods of time, for example, 30 min, 60 min, 1 h, 2 h, 5 h, 10 h, 20 h, 24 h, 48 h, 72 h, and any duration between the listed durations. Preferably, "cultivating for a period of time" refers to cultivating for 24-72 h, for example, 24 h.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 performing homologous recombination repair (HDR).

[0083] This article combines a DNA-dependent protein kinase (DNA-PK) inhibitor and a cell division cycle protein 7 (Cdc7) inhibitor to better improve gene knock-in efficiency in different cell lines, different sites, and different donor template lengths, and is superior to some commercial small molecule enhancers.

[0084] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0085] The embodiments of the present invention are 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 details, and these are all considered to fall within the scope of protection of the present invention.

[0086] Example 1:

[0087] plan:

[0088] Based on relevant literature, we selected five small molecules (the structural formulas are shown below) and tested their effective concentration ranges. Using green fluorescent protein (GFP) as a reporter gene in HEK293T or Jurkat cells, flow cytometry was used to verify the knock-in efficiency of the RAB11A locus at different concentrations of each small molecule, thereby determining the effective concentration of each small molecule.

[0089] method:

[0090] 1) Cell culture

[0091] 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.

[0092] 2) Electroporation

[0093] 2.1) Add a certain amount of culture medium to the well plate (as shown in Table 1)

[0094] Table 1

[0095] 2.2) Add different amounts of small molecules to the culture medium in 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 to preheat.

[0096] Table 2

[0097] Small molecule structure

[0098] Small molecule 1:

[0099] Small molecule 2:

[0100] Small molecule 3:

[0101] Small molecule 4:

[0102] Small molecule 5:

[0103] 2.3) RNP formation: 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) were added to a new, RNase-free centrifuge tube as shown in Table 3 and incubated at room temperature for 5-15 min to form RNPs.

[0104] Table 3

[0105] 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 thoroughly by carefully pipetting with a pipette.

[0106] 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).

[0107] 2.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0108] 2.7) Immediately transfer the electroporated samples to the culture medium in the well plate containing different small molecule concentrations. After electroporation, place the well plate in a 37°C / 5% CO2 incubator for further culture.

[0109] 3) Small molecule mode of action

[0110] Within 72 hours after Neon electroporation, cells were cultured in medium containing small molecules; after 72 hours, the cells were switched from medium containing small molecules to medium without small molecules.

[0111] 4) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect cell viability 3 days after electroporation and the percentage of GFP+ cells (i.e., knock-in efficiency) 7 days after electroporation.

[0112] Experimental conclusion:

[0113] As shown in Figures 1-5, small molecules 1, 2, and 5 significantly improved gene knock-in efficiency, with effective concentrations of: small molecule 1: ≥1 μM; small molecule 2: ≥0.5 μM; and small molecule 5: ≥10 μM. Furthermore, these three small molecules had no significant effect on cell viability within their effective concentrations.

[0114] Example 2:

[0115] plan:

[0116] In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A site was detected by flow cytometry to optimize the incubation conditions of small molecule 5.

[0117] method:

[0118] 1) Different incubation methods of small molecule 5 are shown in FIG6 .

[0119] 2) Cell culture

[0120] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Two aliquots of cells were prepared before electroporation: one aliquot was cultured without small molecule 5, and the other aliquot was treated with small molecule 5 at a final concentration of 30 μM 24 hours prior to electroporation.

[0121] 3) Electroporation

[0122] 3.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

[0123] 3.2) According to the concentration and mode of action of the small molecule in FIG6 , small molecule 5 was added to the culture medium of some wells of the well plate at a final concentration of 30 μM, and then the well plate was placed in a 37° C. incubator for preheating.

[0124] 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 minutes to form RNPs.

[0125] 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 used cells not pre-treated with small molecule 5 in step 2), and the sample group with small molecule 5 used cells pre-treated with 30 μM small molecule 5 in step 2)). Mix carefully by pipetting with a pipette tip.

[0126] 3.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 20 ms, Pulse: 1 pulse.

[0127] 3.6) Use a 10 μl Neon electroporation pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon electroporation pipette tip into a Neon electroporation tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0128] 3.7) Immediately transfer the electroporated samples to the corresponding wells containing medium containing small molecules. After electroporation, place the well plate in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then, transfer the cells to medium without small molecules and culture them.

[0129] 4) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect cell viability 3 days after electroporation and the percentage of GFP+ cells (i.e., knock-in efficiency) 6 days after electroporation (as shown in Figure 7).

[0130] Experimental conclusion:

[0131] As shown in Figure 7, under the premise of no obvious effect on cell viability, the optimal incubation condition of small molecule 5 is incubation method ③: cells are cultured in a culture medium containing small molecule 5 for less than 24 hours before electroporation; after electroporation, cells are cultured in a culture medium containing small molecule 5 for 24 hours. After 24 hours, the cells are transferred from the culture medium containing small molecule 5 to a culture medium not containing small molecule 5.

[0132] Example 3:

[0133] plan:

[0134] In Jurkat cells, five small molecules were used in combination using green fluorescent protein (GFP) as a reporter gene. The knock-in efficiency of the RAB11A site was detected by flow cytometry to confirm the optimal small molecule combination.

[0135] method:

[0136] 1) The effective concentrations and different combinations of small molecules (as shown in Figure 8), where "+" indicates addition and "-" indicates no addition.

[0137] 2) Small molecule incubation time

[0138] Small molecule 1, small molecule 2, small molecule 3, small molecule 4: After electroporation, the cells were cultured in a medium containing the small molecule for 24 hours; after 24 hours, the cells were switched from the medium containing the small molecule to a medium not containing the small molecule.

[0139] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0140] 3) Cell culture

[0141] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Two aliquots of cells were prepared before electroporation: one aliquot was cultured without the addition of the small molecule, and the other aliquot was treated with the small molecule at a final concentration of 30 μM within 24 hours prior to electroporation.

[0142] 4) Electroporation

[0143] 4.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

[0144] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0145] 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 RNPs.

[0146] 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 carefully by pipetting with a pipette tip.

[0147] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 20 ms, Pulse: 1 pulse.

[0148] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0149] 4.7) Immediately transfer the electroporated samples to the corresponding wells containing medium containing small molecules. After electroporation, place the wells in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then transfer the cells to medium without small molecules and culture them.

[0150] 5) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect cell viability 4 days after electroporation (as shown in Figure 9) and the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in Figure 10).

[0151] Experimental conclusion:

[0152] As shown in Figure 10, compared with the use of small molecule 1 alone, the knock-in efficiency of small molecule 1 and small molecule 5 was increased by 20.2%; compared with the use of small molecule 2 alone, the knock-in efficiency of small molecule 2 and small molecule 5 was increased by 22.9%.

[0153] 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.

[0154] Example 4:

[0155] plan:

[0156] In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A site was detected by flow cytometry, further verifying that the two optimal small molecule combinations in Example 3 had a wide applicable concentration range.

[0157] method:

[0158] 1) The effective concentration range of small molecules is shown in FIG11 , where “+” indicates addition and “-” indicates no addition.

[0159] 2) Small molecule incubation time

[0160] Small molecule 1 and small molecule 2: After electroporation, the cells were cultured in a medium containing the small molecule for 24 hours; after 24 hours, the cells were switched from the medium containing the small molecule to a medium without the small molecule.

[0161] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0162] 3) Cell culture

[0163] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Four aliquots were prepared before electroporation: one aliquot was cultured without the small molecule, and the other three aliquots were treated with the small molecule at final concentrations of 20 μM, 30 μM, and 40 μM, respectively, within 24 hours prior to electroporation.

[0164] 4) Electroporation

[0165] 4.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

[0166] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0167] 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 RNPs.

[0168] 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 carefully by pipetting with a pipette tip.

[0169] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1600 V, Width: 10 ms, Pulse: 3 pulses.

[0170] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0171] 4.7) Immediately transfer the electroporated samples to the corresponding wells containing medium containing small molecules. After electroporation, place the wells in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then transfer the cells to medium without small molecules and culture them.

[0172] 5) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in Figure 12).

[0173] Experimental Conclusion

[0174] 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%.

[0175] 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%.

[0176] 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.

[0177] Example 5:

[0178] plan:

[0179] In SJCRH30 cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A site was detected by flow cytometry. Two types of templates, dsDNA and ssDNA, were used to further verify that the combination of these two small molecules has a wide applicability.

[0180] method:

[0181] 1) Small molecule action plan

[0182] 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.

[0183] 2) Small molecule incubation time

[0184] Small molecule 1 and small molecule 2: After electroporation, the cells were cultured in a medium containing the small molecule for 24 hours; after 24 hours, the cells were switched from the medium containing the small molecule to a medium without the small molecule.

[0185] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0186] 3) Cell culture

[0187] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Two aliquots of cells were prepared before electroporation: one aliquot was cultured without the addition of the small molecule, and the other aliquot was treated with the small molecule at a final concentration of 30 μM within 24 hours prior to electroporation.

[0188] 4) Electroporation

[0189] 4.1) Add 1 mL of culture medium (RPMI 1640 containing 10% FBS) to a 12-well plate.

[0190] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0191] 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 minutes to form RNPs.

[0192] Table 4

[0193] 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 carefully by pipetting with a pipette tip.

[0194] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1200 V, Width: 20 ms, Pulse: 3 pulses.

[0195] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0196] 4.7) Immediately transfer the electroporated samples to the corresponding medium containing small molecules. After electroporation, place the well plate in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then transfer the cells to medium without small molecules and culture.

[0197] 5) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in Figure 13).

[0198] Experimental Conclusion

[0199] As shown in Figure 13, for the dsDNA template system, compared with using small molecule 1 or 2 alone, the knock-in efficiency of small molecule 1 or 2 and small molecule 5 in combination increased by 9.85% and 10.9%, respectively; for the ssDNA template system, compared with using small molecule 1 or 2 alone, the knock-in efficiency of small molecule 1 or 2 and small molecule 5 in combination increased by 23.2% and 21.4%, respectively.

[0200] 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.

[0201] Example 6:

[0202] plan:

[0203] In T cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the TRAC site was detected by flow cytometry. Two types of templates, dsDNA and plasmid, were used to further verify that the combination of these two small molecules has a wide applicability.

[0204] method:

[0205] 1) Small molecule action plan

[0206] ① No small molecule; ② 5μM small molecule 1 + 30μM small molecule 5; ③ 2μM small molecule 2 + 30μM small molecule 5.

[0207] 2) Small molecule incubation time

[0208] Small molecule 1 and small molecule 2: After electroporation, the cells were cultured in a medium containing the small molecule for 24 hours; after 24 hours, the cells were switched from the medium containing the small molecule to a medium without the small molecule.

[0209] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0210] 3) Cell isolation, purification, activation and culture

[0211] 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 incubated in a TexMACS containing IL2, IL7, and IL15. TM Culture was performed in Medium (Miltenyi Biotec).

[0212] 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.

[0213] 4) Electroporation

[0214] 4.1) Add 700 μL of culture medium (TexMACS containing 5% FBS, IL2, IL7, and IL15) to a 48-well plate.

[0215] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0216] 4.3) RNP formation: 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) were added to a new, RNase-free centrifuge tube as shown in Table 5 and incubated at room temperature for 5-15 min to form RNPs.

[0217] Table 5

[0218] 4.4) Electroporation system: Add 6 μl of 0.5 M T 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 carefully by pipetting with a pipette tip.

[0219] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 10 ms, Pulse: 3 pulses.

[0220] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0221] 4.7) Immediately transfer the electroporated samples to the corresponding wells containing medium containing small molecules. After electroporation, place the wells in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then transfer the cells to medium without small molecules and culture them.

[0222] 5) Knock-in efficiency detection method: Flow cytometry (Beckman) was used to detect cell viability 3 days after electroporation (as shown in Figure 14) and the percentage of GFP+ cells 7 days after electroporation (i.e., knock-in efficiency, as shown in Figure 15).

[0223] Experimental Conclusion

[0224] 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.

[0225] Example 7:

[0226] plan:

[0227] A 6-bp HindIII restriction endonuclease recognition sequence was inserted into the HPRT locus of Jurkat and SJCRH30 cells, while a 33-bp HiBiT tag was inserted into the VEGFA locus of H1975 and A549 cells. Enzyme digestion, gel analysis, and knock-in efficiency analysis using the Agilent 2100 Bioanalyzer System further validated the broad applicability of these two small molecule combinations in improving knock-in efficiency.

[0228] method:

[0229] 1) Small molecule action plan

[0230] ① No small molecule; ② 5μM small molecule 1 + 30μM small molecule 5; ③ 2μM small molecule 2 + 30μM small molecule 5.

[0231] 2) Small molecule incubation time

[0232] Small molecule 1 and small molecule 2: After electroporation, the cells were cultured in a medium containing the small molecule for 24 hours; after 24 hours, the cells were switched from the medium containing the small molecule to a medium without the small molecule.

[0233] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0234] 3) Cell culture

[0235] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. For each cell type, two aliquots were prepared: one aliquot was cultured without the addition of the small molecule, and the other aliquot was treated with the small molecule at a final concentration of 30 μM within 24 hours prior to electroporation.

[0236] 4) Electroporation

[0237] 4.1) Add a certain amount of culture medium to the well plate (as shown in Table 6):

[0238] Table 6

[0239] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0240] 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 RNPs.

[0241] Table 7

[0242] 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.

[0243] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters as follows: Jurkat: Voltage: 1600 V, Width: 10 ms, Pulse: 3 pulses; SJCRH30: Voltage: 1200 V, Width: 20 ms, Pulse: 3 pulses; H1975: Voltage: 1400 V, Width: 20 ms, Pulse: 2 pulses; A549: Voltage: 1200 V, Width: 20 ms, Pulse: 3 pulses.

[0244] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0245] 4.7) Immediately transfer the electroporated samples to the corresponding wells containing medium containing small molecules. After electroporation, place the wells in a 37°C / 5% CO2 incubator and continue culturing for 24 hours. Then transfer the cells to medium without small molecules and culture them.

[0246] 5) Methods for detecting knock-in efficiency

[0247] 5.1) Three days after electroporation, samples were collected and washed twice with PBS buffer to remove as much PBS as possible from the cells.

[0248] 5.2) Add 50 μl of cell lysis buffer (Lucigen) to the cells, pipette and mix thoroughly, and transfer to a PCR tube. The PCR program is as follows:

[0249] 5.3) 4 μl of the above reaction product was used as a template for PCR amplification in 50 μl of a system using KOD FX Polymerase (TOYOBO) as follows:

[0250] HPRT-F / R and VEGFA-F / R were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0251] PCR procedure:

[0252] 5.4) The above PCR products were recovered by gel (Tian Gen)

[0253] 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 digested with enzymes (HindIII (NEB) for Jurkat and SJCRH30; BsrBI (NEB) for H1975 and A549) using the following enzyme digestion system:

[0254] The enzyme digestion procedure is as follows:

[0255] 5.6) Take 4 μl of the digested product and run it on a 1% agarose gel. The gel image is shown in Figure 16. The digested fragments 1 and 2 are consistent with the theoretical values. Take another 1 μl of the digested product and measure the concentration of each fragment length in the digested product using an Agilent 2100 Bioanalyzer System. The knock-in efficiency is calculated as: (Conc. digested fragment 1 + Conc. digested fragment 2) / (Conc. digested fragment 1 + Conc. digested fragment 2 + Conc. full length) (as shown in Figure 17).

[0256] Experimental Conclusion

[0257] 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).

[0258] In general, both small molecules can greatly improve the efficiency of gene knock-in in different cell lines and different sites, and have wide applicability.

[0259] Example 8:

[0260] plan:

[0261] In Jurkat cells, green fluorescent protein (GFP) was used as a reporter gene, and the knock-in efficiency of the RAB11A site was detected by flow cytometry. The optimized small molecule solutions with higher knock-in efficiency and commercial HDR EnhancerV1 (IDT) for comparison.

[0262] method:

[0263] 1) Small molecule action plan

[0264] 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.

[0265] Commercialized IDT HDR enhancer V1: 30μM (recommended concentration on the IDT official website).

[0266] 2) Small molecule incubation time

[0267] Small molecule 1, small molecule 2, and IDT HDR enhancer: After electroporation, cells were cultured in medium containing the small molecule for 24 h. After 24 h, the cells were switched from medium containing the small molecule to medium without the small molecule.

[0268] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0269] 3) Cell culture

[0270] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Two aliquots of cells were prepared before electroporation: one aliquot was cultured without the addition of the small molecule, and the other aliquot was treated with the small molecule at a final concentration of 30 μM within 24 hours prior to electroporation.

[0271] 4) Electroporation

[0272] 4.1) Add 0.5 mL of culture medium (RPMI 1640 containing 10% FBS) to a 24-well plate.

[0273] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0274] 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 RNPs.

[0275] 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 carefully by pipetting with a pipette tip.

[0276] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1700 V, Width: 20 ms, Pulse: 1 pulse.

[0277] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0278] 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 and continue culturing for 24 hours. Then, transfer the cells to culture medium without small molecules.

[0279] 5) Method for detecting knock-in efficiency: Flow cytometry (Beckman) was used to detect the percentage of GFP+ cells 7 days after electroporation (ie, knock-in efficiency, as shown in FIG18 ).

[0280] Experimental Conclusion

[0281] As shown in Figure 18, compared with the use of small molecule 1 or small molecule 2 alone, the knock-in efficiency of small molecule 1 or 2 and small molecule 5 was increased by about 30%; compared with the IDT HDR enhancer, the knock-in efficiency of small molecule 1 or 2 and small molecule 5 was increased by about 50%.

[0282] Overall, the optimized small molecule combination was superior to the commercial IDT HDR enhancer in improving gene knock-in efficiency.

[0283] Example 9:

[0284] plan:

[0285] 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.

[0286] method:

[0287] 1) Small molecule action plan

[0288] The small molecule combination is: 5 μM small molecule 1 + 30 μM small molecule 5;

[0289] IDT The concentration of HDR enhancer V1 is: 30 μM (recommended concentration on the IDT official website).

[0290] 2) Small molecule incubation time

[0291] 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.

[0292] Small molecule 5: Before electroporation, cells are cultured in a medium containing small molecules for less than 24 hours; after electroporation, cells are cultured in a medium containing small molecules for 24 hours. After 24 hours, cells are transferred from the medium containing small molecules to a medium without small molecules.

[0293] 3) Cell culture

[0294] Cells were passaged every 2-3 days, and cells for electroporation were plated one day prior to electroporation. Two aliquots of cells were prepared before electroporation: one aliquot was cultured without the addition of the small molecule, and the other aliquot was treated with the small molecule at a final concentration of 30 μM within 24 hours prior to electroporation.

[0295] 4) Electroporation

[0296] 4.1) Add 1 mL of culture medium (DMEM containing 10% FBS) to a 12-well plate.

[0297] 4.2) Add different small molecules to the culture medium of some wells of the plate according to the concentration and combination of small molecules, and then place the plate in a 37°C incubator to preheat.

[0298] 4.3) RNP formation: Cas9 protein (Thermo Fisher, A36499), TRAC-sgRNA (SEQ ID NO: 3) (GenScript, EasyEdit), TRAC-86bp dsODN template (annealed product of SEQ ID NO: 4 and 5) (GenScript), and buffer R (Thermo Fisher) were added to a new, RNase-free centrifuge tube as shown in Table 8 and incubated at room temperature for 5-15 min to form RNPs.

[0299] Table 8

[0300] 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 carefully by pipetting with a pipette tip.

[0301] 4.5) Electroporation: Turn on the Neon electroporator (Thermo Fisher) and set the electroporation parameters: Voltage: 1200 V, Width: 10 ms, Pulse: 3 pulses.

[0302] 4.6) Use a 10 μl Neon pipette tip (Thermo Fisher) to aspirate the cell and RNP mixture from the centrifuge tube, ensuring that no bubbles are trapped in the tip. Insert the Neon pipette tip into a Neon tube containing 3 ml of Buffer E (Thermo Fisher). Click the "Start" button on the Neon electroporator screen.

[0303] 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 and continue culturing for 24 hours. Then, transfer the cells to culture medium without small molecules.

[0304] 5) Methods for detecting knock-in efficiency

[0305] 5.1) Take samples 3 days after electroporation and wash them twice with PBS buffer to remove as much PBS buffer as possible.

[0306] 5.2) Add 50 μl of cell lysis buffer (Lucigen) to the cells, pipette and mix thoroughly, and transfer to a PCR tube. The PCR program is as follows:

[0307] 5.3) 4 μl of the above reaction product was used as a template for PCR amplification in 50 μl of a system using KOD FX Polymerase (TOYOBO) as follows:

[0308] TRAC-F and TRAC-R were synthesized at Nanjing GenScript Biotechnology Co., Ltd.

[0309] PCR procedure:

[0310] 5.4) The above PCR products were recovered by gel (Tian Gen)

[0311] 5.5) The concentration of the gel-recovered product was determined using a Nanodrop assay. 200 ng of the gel-recovered product was digested with HindIII (NEB) using the following enzyme digestion system:

[0312] The enzyme digestion procedure is as follows:

[0313] 5.6) Take 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 knock-in efficiency is calculated as follows: (Conc. digested fragment 1 + Conc. digested fragment 2) / (Conc. digested fragment 1 + Conc. digested fragment 2 + Conc. full length) (as shown in Figure 19).

[0314] Experimental Conclusion

[0315] As shown in Figure 19, the combination of small molecules 1 and 5 increased knock-in efficiency by 31.9% compared to the IDT HDR enhancer. At the TRAC site in HEK293T cells, the optimized small molecule combination was superior to the commercial IDT HDR enhancer in improving knock-in efficiency.

[0316] The CRISPR / Cas9 system is a widely used genome editing tool that enables precise gene editing at specific sites. Although many small molecules have been screened and discovered to improve the efficiency of precise gene editing by regulating the selection of cellular repair pathways, these small molecules have certain cell type specificity and context dependence, and the degree of improvement described by different authors also varies. The present invention uses two small molecule compounds in combination to provide a method with a wide range of applications that can significantly improve the efficiency of CRISPR / Cas9-mediated precise gene editing.

[0317] The present invention uses two small molecule compounds in combination to provide a method for improving the efficiency of CRISPR / Cas9-mediated precise gene editing, comprising:

[0318] Targeting different target sites in different cells, verify the knock-in efficiency of three small molecules, small molecule 1, small molecule 2, and small molecule 5, at different concentrations to determine the optimal concentration of each small molecule for that cell line and site.

[0319] 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.

[0320] 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.

[0321] The nucleotide sequences mentioned in this article are as follows:

Claims

1. A method for enhancing the efficiency of gene knock-in mediated by the CRISPR gene editing system in cells, comprising contacting the cells with a DNA-dependent protein kinase inhibitor and a cell division cycle protein 7 inhibitor during the gene knock-in operation on the cells.

2. The method of claim 1, wherein the CRISPR gene editing system comprises a Cas9 protein.

3. 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.

4. The method of any one of claims 1 to 3, wherein the donor template is a DNA molecule in single-stranded or double-stranded form.

5. The method according to any one of claims 1 to 4, wherein the DNA-dependent protein kinase inhibitor is selected from the group consisting of 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:

6. The method according to any one of claims 1 to 5, wherein the cyclin 7 inhibitor is a compound of formula (V) or its hydrochloride:

7. 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.

8. The method of 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.

9. The method of any one of claims 5 to 8, wherein: 1) contacting the cells with the compound of formula (I) at a concentration of 5-20 μM, and contacting the cells with the compound of formula (V) 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.

10. 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.

11. The method of any one of claims 1 to 10, wherein the cell is a HEK293T, Jurkat, SJCRH30, H1975, or A549 cell.

12. The method of any one of claims 1-11, wherein the cell is a T cell.

13. The method of 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 a gene knock-in procedure.

14. The method of 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) prior to the transfection.

15. 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) are 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.

16. A kit for enhancing the efficiency of gene knock-in mediated by the CRISPR gene editing system in cells, comprising a DNA-dependent protein kinase inhibitor and a cell division cycle protein 7 inhibitor.

17. The kit of claim 16, wherein the kit further comprises a Cas9 protein or a nucleic acid molecule encoding the Cas9 protein.

18. The kit of claim 16 or 17, wherein the kit further comprises a guide RNA and / or a donor template for homologous recombination repair.

19. 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.

20. The kit according to any one of claims 16 to 19, wherein the DNA-dependent protein kinase inhibitor is selected from the group consisting of 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:

21. The kit according to any one of claims 16 to 20, wherein the cyclin 7 inhibitor is a compound of formula (V) or its hydrochloride:

22. The kit of any one of claims 16 to 21, wherein the cells are HEK293T, Jurkat, SJCRH30, H1975, or A549 cells.

23. The kit of any one of claims 16-22, wherein the cells are T cells.

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