CN110241136B - Small molecule compound for promoting gene editing and application thereof - Google Patents

Abstract

The invention provides a small molecule compound for promoting gene editing and application thereof. Specifically, the invention provides application of a compound shown as a formula A or a pharmaceutically acceptable salt thereof, which are used for preparing an accelerating agent or a preparation for accelerating gene editing, wherein the structure of the formula A is described in the specification. The compound can obviously improve the efficiency of CRISPR gene editing, thereby providing a simple and efficient strategy for accurate gene editing and providing a new method and a new tool for genome engineering.

Description

Small molecule compound for promoting gene editing and application thereof

Technical Field

The invention relates to the field of biology, in particular to a small molecule compound for promoting gene editing and application thereof.

Background

With the advent of gene editing technology, efficient gene editing and modification for different cells has become possible. Taking pluripotent stem cells as an example, the pluripotent stem cells comprise embryonic stem cells (hESCs) and induced pluripotent stem cells (hipSCs), and can be used for researching early development and disease occurrence and development. Therefore, it is crucial to perform rapid, efficient, and controllable gene editing on cells (including somatic cells, pluripotent stem cells, etc.).

The site-specifically recognized nuclease can cause DNA double strand breaks at specific locations in the genome and trigger endogenous DNA repair mechanisms. Repair of DNA double strand breaks (NHEJ) by non-homologous end joining approaches, which can be used to generate knock-out mutants, results in small fragment insertions or deletions. Homology Directed Repair (HDR) can then be used to construct knock-in mutants or reporter cell lines. However, even with the assistance of these site-specific nucleases, precise genome editing by homology-directed repair remains very challenging.

With the discovery of nucleases such as Cas9, several CRISPR technology-based gene editing techniques have been developed, such as CRISPR-Cas 9-mediated gene editing.

Some small molecule compounds have been found to modulate CRISPR-Cas9 mediated gene editing processes. Yu et al found that two small molecules, L755507 and Brefeldin A, can promote CRISPR-Cas9 mediated homology directed repair. Chu and Maruyama et al found that SCR7, an inhibitor of ligase IV, can increase CRISPR-Cas 9-mediated gene editing efficiency.

The CRISPR-Cpf1 nuclease-based gene editing technology is another directional gene editing technology, the range of gene editing is expanded, and higher precision is achieved, however, the CRISPR-Cpf1 gene editing efficiency is still unsatisfactory. In addition, chemical small molecules that can facilitate CRISPR-Cpf1 gene editing have not been reported.

Therefore, there is an urgent need in the art to develop new compounds that are effective in improving the efficiency of gene editing.

Disclosure of Invention

The invention aims to provide a compound capable of effectively improving gene editing efficiency and application thereof.

In a first aspect of the invention, the invention provides a compound shown in formula a, or a pharmaceutically acceptable salt thereof, or an optical isomer or a racemate thereof, or a solvate thereof, for use in preparing an accelerating agent or a preparation for accelerating gene editing;

in the formula (I), the compound is shown in the specification,

each R1 is independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl;

n is 0, 1, 2, 3, or 4;

r2 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl;

r3 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl;

r4 is selected from the group consisting of: H. halogen, - (L1) p-substituted or unsubstituted C1-C6 alkyl, - (L1) p-N (Ra) (Rb), - (L1) p substituted or unsubstituted C2-C6 alkenyl, - (L1) p-substituted or unsubstituted C3-C6 cycloalkyl;

r6 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl, - (L2) q-substituted or unsubstituted C1-C6 alkyl;

wherein p is 0, 1, 2, or 3; and each L1 is independently selected from the group consisting of: -CH ₂ -, -NH-, -S-, -O-, or combinations thereof;

in another preferred embodiment, R6 is selected from the group consisting of: halogen, C1-C6 alkyl or halogenated C1-C6 alkyl.

In another preferred embodiment, R6 is F, cl, or Br.

In another preferred embodiment, the compound is a compound of formula A1:

in another preferred embodiment, the compound is

In another preferred embodiment, R3 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 cycloalkyl;

in another preferred embodiment, R4 is selected from the group consisting of: H. halogen, - (L1) p-substituted or unsubstituted C1-C6 alkyl, - (L1) p-N (Ra) (Rb), - (L1) p substituted or unsubstituted C2-C6 alkenyl, - (L1) p-substituted or unsubstituted C3-C6 cycloalkyl;

in another preferred embodiment, p is 0, 1, or 2;

in another preferred embodiment, each L1 is independently selected from the group consisting of: -CH ₂ -, -NH-, -O-, or combinations thereof;

in another preferred embodiment, said gene editing comprises CRISPR-based gene editing.

In another preferred example, the gene editing comprises CRISPR-Cpf 1-based gene editing and CRISPR-Cas 9-based gene editing;

in another preferred embodiment, said gene editing comprises in vivo gene editing, in vitro gene editing, or a combination thereof;

in another preferred embodiment, the sample against which the gene editing is directed is selected from the group consisting of: a cell, tissue, organ, or combination thereof;

in another preferred embodiment, the sample is from animals, plants, microorganisms (including bacteria and viruses);

in another preferred embodiment, the sample is from a human or non-human mammal;

in another preferred embodiment, the cells include primary cells and passaged cells;

in another preferred embodiment, the cells include somatic cells, germ cells, stem cells;

in another preferred embodiment, the stem cells comprise: pluripotent, multipotent, and multipotent stem cells;

in another preferred embodiment, the stem cells are induced pluripotent stem cells (hipscs);

in another preferred embodiment, the cell comprises: embryonic stem cells, adipose stem cells, hematopoietic stem cells, immune cells (e.g., T cells, NK cells);

in another preferred embodiment, the formulation comprises a pharmaceutical composition.

In a second aspect of the invention, there is provided a method of promoting gene editing in a cell in vitro, the method comprising:

(a) (ii) performing gene editing on the cell in the presence of a gene editing-promoting agent, thereby promoting gene editing in the cell,

wherein the gene editing promoter is a compound shown in formula A, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; (wherein formula a is as defined in claim 1);

in another preferred example, in step (a), the gene editing promoter is contacted with the cell undergoing gene editing before, during and/or after gene editing has been performed;

in another preferred embodiment, said in vitro gene editing is performed in an in vitro reaction system;

in another preferred embodiment, in the in vitro reaction system, the concentration of the gene editing promoter is 0-2 μ M;

in another preferred example, in the step (a), the method comprises:

(a1) Introducing into the cell a nucleotide sequence encoding a Cpf1 nuclease, wherein the Cpf1 nuclease is capable of generating a double-strand break in the target DNA, thereby inducing gene editing of the target DNA; and

(a2) Culturing said cell in the presence of said gene-editing promoting agent;

in another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a third aspect of the invention, a reagent product (or combination of reagents) comprises:

(i) A first reagent which is a gene editing accelerator and is a compound shown in a formula A, or pharmaceutically acceptable salt, an optical isomer, a racemate or a solvate of the optical isomer; (wherein formula a is as defined in claim 1); and

(ii) A second agent that is an agent that performs CRISPR gene editing;

in another preferred embodiment, the second agent comprises one or more agents selected from the group consisting of:

(c1) A Cpf1 nuclease, a coding sequence for a Cpf1 nuclease, or a vector expressing a Cpf1 nuclease, or a combination;

(c2) A Cas9 nuclease, a coding sequence for a Cas9 nuclease, or a vector expressing a Cas9 nuclease, or a combination;

(c3) A crRNA, a vector for producing the crRNA;

(c4) Template for homogenous directed repair: a single-stranded nucleotide sequence or a plasmid vector.

In a fourth aspect of the invention, there is provided a kit for preparing for gene editing;

in another preferred embodiment, the kit further comprises instructions;

in another preferred embodiment, the specification describes a method for promoting gene editing according to the present invention;

in another preferred embodiment, the gene editing is gene editing for somatic cells and stem cells;

in another preferred example, the gene editing is CRISPR-Cpf 1-based gene editing;

in another preferred embodiment, the cell is selected from the group consisting of: embryonic stem cells, induced pluripotent stem cells, human embryonic kidney 293T cells;

in another preferred embodiment, the gene editing is directed to a disease-causing gene, a tumor-associated gene (e.g., an oncogene), an immune-associated gene (e.g., a gene associated with autoimmunity), a vision-associated gene;

in another preferred embodiment, the gene is selected from the group consisting of: OCT4, ALBUMIN, ALKBH1 or combinations thereof.

In a fifth aspect of the invention, there is provided a kit comprising:

(i) The reagent comprises a first container and a first reagent positioned in the first container, wherein the first reagent is a gene editing accelerator, and the gene editing accelerator is a compound shown as a formula A, or pharmaceutically acceptable salt, optical isomer, racemate or solvate thereof; (wherein formula a is as defined in claim 1); and

(ii) A second container, and a second agent located within the second container, the second agent being an agent that performs CRISPR gene editing.

In a sixth aspect of the present invention, there is provided a method of facilitating gene editing, comprising the steps of: administering a gene editing promoter and a gene editing reagent for gene editing to a subject in need thereof, wherein the gene editing promoter is a compound represented by formula A, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof; (wherein formula a is as defined in claim 1);

in another preferred embodiment, the subject includes human and non-human mammals;

in another preferred embodiment, said gene editing reagent comprises a CRISPR-Cpf 1-based gene editing reagent.

In another preferred embodiment, a gene-editing reagent that effects gene editing is administered to the subject before, during and/or after administration of the gene-editing promoting agent.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 shows that CRISPR-Cpf1 efficiently generated knockout hPSC cell lines.

Figure 2 shows the identification of small molecules capable of significantly facilitating CRISPR-Cpf 1-mediated knock-in of genes in human pluripotent stem cells.

Fig. 3 shows the establishment of small molecules that significantly facilitate CRISPR-Cpf 1-mediated knock-in of hPSC gene into cell lines.

Figure 4 shows a working model of CRISPR-Cpf 1.

FIG. 5 shows an identification model for CRISPR-Cpf1 generation knock-out hPSC cell lines.

Fig. 6, 7 show potential off-target site analysis for CRISPR-Cpf 1.

FIG. 8 shows that the candidate small molecule can significantly promote CRISPR-Cpf1 or CRISPR-Cas9 mediated hPSC gene knock-in at OCT4 site.

Figure 9 shows toxicity testing of candidate small molecules.

Figure 10 shows that candidate small molecules can significantly promote CRISPR-Cpf1 or CRISPR-Cas9 mediated knockin of hPSC genes at the ALBUMIN site.

Fig. 11 shows that the candidate small molecules can significantly promote CRISPR-Cpf 1-mediated knockin of hPSC double genes.

Detailed Description

The inventor of the invention, after extensive and intensive research, unexpectedly discovers that a compound with a structure shown as formula A can significantly improve the gene editing efficiency of CRISPR, and especially can significantly promote the gene knock-in efficiency mediated by CRISPR-Cpf 1. Experiments show that the compound of the formula A (such as AZD-7762) can remarkably promote CRISPR-Cpf 1-mediated gene editing efficiency, for example, efficient gene editing is carried out on human pluripotent stem cells so as to generate a gene knock-in cell line. On the basis of this, the inventors have completed the present invention.

Term(s) for

The term "C ₁ -C ₆ Alkyl "means a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or the like.

The term "C ₃ -C ₆ Cycloalkyl "refers to cycloalkyl groups having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or the like.

The term "C ₁ -C ₃ Alkylene "means a straight or branched chain alkylene group having 1 to 3 carbon atoms, such as methylene, ethylene, propylene, or the like.

The term "C5-C7 cycloalkyl" refers to a cycloalkyl group having 5 to 7 carbon atoms, such as cyclopentyl, cyclohexyl, or the like.

The term "C ₁ -C ₆ Haloalkyl "refers to a halogen-substituted straight or branched chain alkyl group having 1 to 6 carbon atoms, such as halomethyl, haloethyl, halopropyl, haloisopropyl, halobutyl, haloisobutyl, or the like.

The term "halogen" refers to F, cl, br and I.

Gene editing promoter

As used herein, "the compound of the present invention", "the compound of formula a", "the gene editing promoter of the present invention" are used interchangeably and refer to a compound having the structure represented by formula a, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof. It is to be understood that the term also includes mixtures of the above components.

In the formula, each group is as defined above.

The compound has extremely obvious promotion effect on the efficiency of gene editing

In the present invention, pharmaceutically acceptable salts of the compounds of formula a are also included. The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed with acids from the compounds of the present invention. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

The compounds of formula a of the present invention can be prepared by methods well known to those skilled in the art, and the reaction parameters of the respective steps are not particularly limited.

As used herein, in the compound of formula a, if a chiral carbon atom is present, the chiral carbon atom may be in the R configuration, may also be in the S configuration, or a mixture of both.

Gene editing

The compound can obviously improve the efficiency of gene editing.

In the present invention, representative gene edits include (but are not limited to): CRISPR-based gene editing. Typically, CRISPR-based gene editing includes CRISPR-Cpf 1-based gene editing, CRISPR-Cas-based gene editing. Wherein, the Cas includes Cas9 and the like.

One preferred gene editing is CRISPR-Cpf1 gene editing. The CRISPR-Cpf1 can specifically recognize a thymine nucleotide (T) -rich PAM sequence (TTTN), and expands the range of gene editing mediated by RNA. CRISPR-Cpf1 generates a 5nt sticky end that initiates a different DNA repair process. The crRNA length of CRISPR-Cpf1 is much shorter than that of Cas9, and in vitro synthesis is easier and more suitable for polygene editing process. The target miss rate of CRISPR-Cpf1 is also low.

Use of

The combination of the compound of formula a and a gene editing reagent (such as a CRISPR-Cpf1 reagent) of the present invention can significantly improve the efficiency of gene editing, and thus has revolutionary potential in different fields such as therapeutic applications.

The compound of the formula A can be used for improving the CRISPR-mediated gene editing efficiency, and further can be used for preventing or treating diseases related to pathogenic genes.

In one embodiment, the invention provides an in vitro non-therapeutic small molecule screening method for promoting CRISPR-mediated gene editing comprising: CRISPR-mediated gene knock-in and drug screening systems.

The invention also provides a method of using small molecules to facilitate CRISPR-mediated gene editing, which method may be therapeutic or non-therapeutic. Generally, the method comprises the steps of: administering to a subject in need thereof a compound of formula a of the present invention.

Preferably, the subject includes human and non-human mammals (rodents, rabbits, monkeys, domestic animals, dogs, cats, etc.).

Compositions and methods of administration

The present invention provides a composition for promoting CRISPR-mediated gene editing efficiency. The composition includes (but is not limited to): pharmaceutical compositions, scientific research reagent compositions, and the like.

In the present invention, the composition can be directly used to promote gene editing, for example, single gene knock-in, double gene knock-in, point mutation, etc.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of a compound of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration.

In the case of pharmaceutical compositions, the compositions of the present invention may be prepared in the form of injections, for example, by conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for aerosol inhalation.

For the pharmaceutical compositions of the present invention, administration to a subject in need thereof (e.g., human and non-human mammals) can be carried out by conventional means. Representative modes of administration include (but are not limited to): oral, injection, topical administration, and the like.

The main advantages of the invention include:

(a) The small molecule compound which can remarkably promote gene editing is provided for the first time, and the compound is particularly effective to CRISPR-Cpf1/Cas9 mediated hPSC gene knock-in editing.

(b) The invention provides a simple and efficient strategy for precise gene editing based on the combination of the compound shown in the formula A and the CRISPR-Cpf 1.

(c) Based on the high-throughput screening method of the pluripotent stem cells, an unbiased drug screening system is established.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

General procedure

Plasmid construction

For the pCpfcr vector, the BbsI enzyme recognition sequence and DR (forward repeat) sequence were designed in the upstream primer of the U6 promoter; the U6 promoter region was amplified by PCR using pUcm-T vector cloning kit (Sangon Biotech Co.) and then cloned into T vector. To construct the pCpfcr-crRNA vector to express the targeting sequence of Cpf1 (crRNA), a 24bp oligonucleotide was first synthesized based on the crRNA sequence, annealed to form a double-stranded DNA fragment, and then cloned into the BbsI-digested pCpfcr vector.

For construction of OCT4-tdTomato donor Plasmid, mOrange sequence was removed from OCT4-2A-mOrange donor Plasmid (Addge, plasmid # 66986) as backbone by PCR, followed by PCR amplification of tdTomato sequence, which was ligated to the backbone using Gibson Assembly kit (New England Biolabs). All vectors were checked by Sanger sequencing.

Cell culture

hPSCs were cultured according to a conventional method. The H1 embryonic stem cell line, the MEL1 embryonic stem cell line and the hipSCs are cultured by an hPSC culture medium, and the hPSC culture medium comprises the following components: DMEM/F12 (Life Technologies), 20% serum replacement KSR (Life Technologies), 1 Xnon-essential amino acids (Life Technologies), 100 Xpenicillin/streptomycin (Life Technologies), 0.055mM mercaptoethanol (Sigma), and 10ng/mL bFGF (PeProtech).

Every 3-6 days, hPSC cells were digested with Accutase (Life Technologies) and passaged 1:3 to 1:6. 0.5. Mu.M Thiazovivin was added to the hPSC medium at each passage or thawing of the cells.

Electric converter

Plasmids were extracted using a ZymoPURE plasmid extraction kit and then subjected to electroporation. Firstly, 81.82 μ L Solution I and 18,18 μ L Supplement I (Human Stem Cell) are taken

Kit 1, lonza) and plasmid were mixed together to make electrotransfer solution.

For the knock-in experiments, the plasmid consisted of 3. Mu.g of pcDNA3.1-hLbCpf1 (Addgene, plasmid # 69988), 3. Mu.g of pCpfcr-crRNA and 4. Mu.g of OCT4-tdTomato donor OCT4-2A-eGFP-PGK-Puro donor (Addgene, plasmid # 31938). hPSC cells were digested into single cells with Accutase, 1X10 cells were collected ⁶ Individual cells were resuspended using electrotransfer solution and electrotransferred using Amaxa electrotransfer (Lonza). After electroporation, cells were plated in one well of a six-well plate for 2-3 days. Afterwards, knock-in efficiency was analyzed by flow or T7 EI.

For the establishment of cloning experiments, 500-2000 cells were seeded into 10cm dishes and cultured for 4-7 days. Clones were picked for amplification and storage. After 2 weeks, cell lines expressing tdTomato were identified by immunostaining, flow, sequencing, etc.

Transfection

Human embryonic kidney 293T cells were cultured in DMEM (Life Technologies) plus 10% fetal bovine serum, 100 XPicillin/streptomycin medium. When the 293T cell density reaches 70% -80%, lipofectamine is used ^TM 3000 transfection reagent (Invitrogen) was mixed with the plasmid for transfection. The plasmid mixture consisted of 1. Mu.g of pY016 (pcDNA3.1-hLbCpf 1) and 1. Mu.g of pCpfcr-crRNA. After 2-3 days of transfection, the cells were analyzed by T7EI assay.

PFLP and T7EI assays

Cells were harvested 2-3 days after transfection or electroporation and the genome was extracted using a Rapid genomic DNA miniprep kit (ZYMO). And amplifying the fragment with the CRISPR action target on the genome by using Taq polymerase (Vazyme).

For RFLP experiments, 2. Mu.L of the amplification product was digested with restriction enzymes and analyzed by running electrophoresis on a 2% agarose gel.

For the T7EI experiment, 10. Mu.L of the amplified product was mixed with NEB buffer 2 (New England Biolabs) to prepare a total volume of 16. Mu.L. The mixed system was incubated at 95 ℃ for 5 minutes, then at 2 ℃ per second to 85 ℃, then at 0.1 ℃ per second slowly to 25 ℃, and finally at 4 ℃. To 16. Mu.L of the reaction product, T7EI enzyme (New England Biolabs) was added to prepare a total volume of 20. Mu.L, and the system was incubated at 37 ℃ for 30 minutes, followed by running electrophoresis on 2.5% agarose gel. The Gel images were taken by JS-2000Gel Imager (Peiqing Science & Technology) and analyzed by ImageJ software.

Mutation rate was determined by the formula 100 × (1- (1- (b + c)/(a + b + c)) ^1/2 ) And calculating, wherein a represents the band intensity of the product which is not digested by T7EI, and b and c respectively represent the band intensity of the product after digestion.

Flow cytometry

hPSCs transfected with Cpf1, OCT4-crRNA and OCT4-tdTomato donor were seeded in one well of six-well plates and cultured in hPSC medium for 3-4 days. Differentiated and undifferentiated cells were digested with Accutase for 3-5 minutes into single cells. Then resuspended in 0.3-1mL 1 XPhosphate buffer. And finally, obtaining data by using a Beckman flow cytometer, and analyzing the result.

hPSC differentiation

Approximately 200 undifferentiated OCT4-tdTomato hPSC cells were seeded in one well of a six-well plate and cultured with hPSC medium for 4-7 days. Then, the cells were cultured in a differentiation medium (DMEM/F12, 20% serum replacement, 1 XNEAA, 100 Xpenicillin/streptomycin (Life TechniLOgies), 0.055mM 2-mercaptoethanol (Sigma), 0.1. Mu.M LDN225, 10. Mu. M E616452) for 3 days. After the fourth or fifth day, the cells can be analyzed by flow and immunostaining.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 10-15 minutes at room temperature followed by three washes of 1 XPBST (1XPBS +0.3% Triton X-100 (Vetec)) for five minutes each. Next, after the cells were incubated in blocking solution (1x PBST +5% BSA (fetal bovine serum)) for 0.5-1 hour at room temperature, primary antibody was incubated overnight (4 ℃). Subsequently, PBST was washed three times for 15 minutes each at room temperature, and then the corresponding secondary antibody was added and incubated for 1 hour. Finally, nuclei were stained using Hoechst (1.

Apoptosis assay

Apoptosis was analyzed by an apoptosis kit. Human pluripotent stem cells were first treated with DMSO, AZD-7762 (1. Mu.M) and VE-822 (1. Mu.M). Collection of 5X10 ⁵ Cells were washed twice with ice PBS. Cells were centrifuged at 4 ℃ for 5 minutes, then resuspended with 50. Mu.L of 1 × Binding Buffer, and 2.5. Mu.L of Annexin V (AV) -FITC and 2.5. Mu.L of PI staining solution were added and incubated at room temperature for 10-15 minutes. Then 250. Mu.L of 1 × Binding Buffer was added to the mixture. And finally detecting apoptosis by using a flow cytometer.

Off-target analysis

Cas-OFFinder can be used to detect potential off-target sites. The mismatch value is set to 6 or less. After potential off-target sites are found, primers are designed, PCR is used for amplifying off-target site fragments on a cell genome, and sequencing identification is carried out through a Sanger sequencing method.

Statistical analysis

The two-tailed t-test gives P-values and all statistics are expressed as mean ± s.e.

Example 1.

Efficient construction of gene knockout cell lines using CRISPR-Cpf1 in human pluripotent stem cells

To use the CRISPR-Cpf1 gene editing system in human pluripotent stem cells, a crRNA expression plasmid was constructed that was driven by the U6 promoter (fig. 4a and 4 b).

Several genes of interest were selected, including ALKBH1 and CLEC16A. ALKBH1 is a tRNA demethylase, and CLEC16A plays an important role in the development of diabetes.

To construct gene-specific crRNA plasmids, a set of crRNAs was designed that specifically target these genes (fig. 1b and table 1).

[ TABLE 1 ]

Construction of nucleic acid sequences for expression of crRNA plasmids

PCR primers for plasmid construction

PCR primers for genotyping and sequencing

PCR primer for T7EI experiment

Primer for RFLP experiment

Primers for qPCR

PCR primers for off-target rate analysis

ssODN template sequences

These crRNAs were tested for genome editing capacity in 293T cells. In the T7E1 (T7 endonuclease I) assay, a 20-30% indel ratio was observed (FIG. 4 c).

The ability of CRISPER/Cpf1 mediated genome editing in hPSCs was then investigated (FIG. 1 a). In the T7E1 assay, 20-30% efficiency was also observed in the gene knock-outs for ALKBH1 and CLEC16A in hESCs and hipSCs (FIG. 1 c).

To further establish knock-out hPSC lines, we passaged transfected hPSCs at low cell density, picked clones, and analyzed by PCR genotyping (fig. 1 a).

For ALKBH1, single allele knock-outs occurred in 20.8% of clones and double allele knock-outs occurred in 12.5% (FIG. 1 e).

For CLEC16A, single allele knockdown occurred in 25% of clones, and double allele knockdown occurred in 16.7% of clones (fig. 1 e).

The sanger sequencing result proves that after gene knockout occurs on ALKBH1 and CLEC16A targeting sites, a genome sequence lacks part of basic groups, so that the gene knockout is realized (FIG. 1 f).

In addition, some potential off-target sites found based on the software analysis were also analyzed by PCR amplification and sanger sequencing, and no off-target phenomenon was found (fig. 6, 7 b).

In conclusion, the above experimental results clearly demonstrate that CRISPR-Cpf1 can efficiently knock out genes in hPSCs.

Example 2.

Screening for small molecules that can significantly promote CRISPR-Cpf 1-mediated HDR in hPSCs

In this example, to examine the ability of CRISPR-Cpf1 to perform gene insertion, three plasmids were transferred into hPSCs using the transfer method: one plasmid expresses Cpf1, one contains specific crRNA targeting OCT4, and HDR template plasmid containing eGFP reporter gene and puromycin (Puro) resistance (fig. 8 a). Following electroporation, hPSCs were cultured in normal medium for two days, after which puromycin was added to the medium for 3-4 days. The efficiency of genome repair by HDR means is observed to be relatively low and needs to be further improved.

In addition, SCR7 compounds known to increase CRISPR-Cas9 mediated gene editing efficiency were employed as control compounds.

As a result:

experiments show that SCR7, a small molecule known to promote CRISPR-Cas 9-mediated gene knock-in, has no significant promoting effect in CRISPR-Cpf 1-mediated gene knock-in (fig. 2 e).

This indicates that there are some differences between the CRISPR-Cpf1 system and the CRISPR-Cas9 system in practical applications. Therefore, it is important to find new small molecule compounds that promote HDR effectively.

Example 3

Chemical small molecule screening

In this example, screening and testing were performed one by one for hundreds of candidate compounds.

In this example, to increase the efficiency of CRISPR-Cpf 1-mediated gene editing in hPSCs, large-scale compound screening was performed using the OCT4-eGFP knock-in screening system with puromycin (fig. 2 a).

Specifically, three plasmids were transferred into hPSCs using the electrotransfer method: one plasmid expresses Cpf1, one contains specific crRNA targeting OCT4, and HDR template plasmid containing eGFP reporter gene and puromycin (Puro) resistance (fig. 8 a).

For each candidate compound, 100 μ L of cell culture medium and 0.2 μ L of the selected small molecule (1. After electroporation of the cells, 1X10 ⁶ The cells were mixed well in 4.8mL of hPSC medium supplemented with Thiazovivin small molecules and plated in a 48-well plate. Culturing cells by using a culture medium added with AZD-7762 micromolecules, stopping adding the AZD-7762 micromolecules after two days, adding 1 mu g/mL puromycin in the culture medium, and continuing culturing for 3-5 days. Subsequently, the efficiency of the small molecules was tested by counting the clones selected to have puromycin resistance.

As a result:

in total, about 600 small molecules of the kinase library were screened, with the small molecule AZD-7762 very significantly increasing the number of hPSC clones (FIGS. 2b,2c,2e,2f,3b,3c,8d,10b,10e and 11 a), suggesting that the compound AZD-7762 may significantly increase gene editing efficiency.

Example 4

Effect verification of Gene editing promoters

In this example, further experiments were carried out to verify the effect of AZD-7762. The test method was the same as in example 3.

The results show that AZD-7762 achieves its maximum effect at 1. Mu.M (FIG. 2 d). AZD-7762 is an inhibitor of ATR kinase, and can improve gene knock-in efficiency by 2.7 times.

Furthermore, AZD-7762 did not significantly increase the cell proliferation rate of hPSCs, indicating that AZD-7762 did not increase the efficiency of gene knock-in by promoting cell proliferation (FIG. 9 a).

Furthermore, AZD-7762 did not exhibit cytotoxicity against hPSCs (FIGS. 9c and 9 d). hPSCs treated with AZD-7762 still highly expressed specific pluripotency genes for OCT4 and NANOG (FIG. 9 b).

In contrast, the control compound SCR7 did not significantly promote CRISPR-Cpf 1-mediated knock-in hPSCs, but AZD-7762 could significantly promote Cpf 1-mediated knock-in hPSCs (fig. 2e and 8 d).

In the CRISPR-Cas9 system, the SCR7 improved efficiency by less than 2-fold, while the effect of AZD-7762 was more pronounced (about 3-fold).

Furthermore, knocking down the CHEK1 gene with siRNAs could promote CRISPR-Cpf1 mediated gene knock-in efficiency, suggesting that AZD-7762 does act by targeting CHEK1 (fig. 2 g). The results successfully confirm that AZD-7762 can significantly improve CRISPR-Cpf1 mediated gene knock-in efficiency in hPSCs.

Example 5.

Small molecules significantly promote CRISPR-Cpf 1-mediated generation of hPSCs gene knock-in cell lines

In this example, the ability of CRISPR-Cpf1 to generate hPSCs knock-in cell lines without drug screening was further examined.

The method comprises the following steps: 3 plasmids were electrotransferred into hPSCs: one plasmid expresses Cpf1, one contains crRNA specifically targeting OCT4, and one donor plasmid containing OCT4-2A-tdTomato (fig. 3 a). After 5-6 days, we observed tdTomato positive cells, and examined the efficiency of gene knock-in by FACS (flow analysis).

The results show that: AZD-7762 was able to significantly increase the percentage of tdTomato positive cells, indicating that AZD-7762 could promote CRISPR-Cpf1 knock-in hPSCs (FIG. 3b, c).

Thereafter, several tdTomato positive clones were picked for establishment of hPSC cell lines. The results of PCR analysis showed successful integration of the tdTomato reporter in these cell lines (fig. 3 f). These established hPSC cell lines co-expressed tdTomato and pluripotency gene OCT4 (fig. 3 d).

After differentiation of these stem cells, it was confirmed by immunostaining and FACS experiments that the cells lost the expression of the pluripotent gene OCT4, and at the same time, the cells no longer expressed the tdTomato fluorescent reporter (FIG. 3d, e). Therefore, OCT4-tdTomato can synchronously reflect the expression of the endogenous pluripotency gene OCT4 during the hPSCs stage and differentiation.

Thereafter, the sequencing was performed by the sanger method. Sequencing results suggest that in these OCT4-tdTomato cell lines, the 2A-tdTomato sequence was successfully inserted into the genome and replaced the stop codon sequence of the OCT4 gene.

In addition, the double-gene knock-in experiment performed on the two gene loci of OCT4 and ALBUMIN proves that AZD-7762 can remarkably promote the efficiency of CRISPR-Cpf 1-mediated double-gene knock-in (figure 11 a).

For the gene point mutation experiment (FIG. 3 h) using CRISPR-Cpf1 mediated generation, the method is to electrotransfer the vector for expressing Cpf1, the ALKBH1-crRNA vector and the single-stranded nucleic acid sequence template into hPSC cells. The results show that the candidate small molecule combination can significantly promote the efficiency of constructing gene point mutation by using single-stranded nucleic acid as a template (fig. 3i and 3 j).

The above experimental results show that efficient gene insertion in hPSCs can be performed based on a combination of CRISPR-Cpf1 and small molecules.

Discussion of the preferred embodiments

Experiments prove that efficient gene knockout and insertion editing can be performed in a human pluripotent stem cell line based on CRISPR-Cpf1 gene editing. The T7EI experiment result proves that CRISPR-Cpf1 successfully performs genome cutting on specific sites of two genes of ALKBH1 and CLEC16A. And then, the genotype identification is carried out by a monoclonal picking method, and the result shows that the gene knockout efficiency is higher.

The sequencing result also can conclude that the CRISPR-Cpf1 can successfully construct a gene knockout cell line.

For gene insertion experiments, the inventor constructs a vector of OCT4-tdTomato, successfully inserts a red fluorescent reporter gene into the OCT4 gene through a CRISPR-Cpf1 mediated gene editing technology, and can detect hPSCs successfully inserted with the fluorescent reporter gene through flow cytometry analysis and immunofluorescence staining experiments.

In addition, the CRISPR-Cpf1 is unique in that the crRNA is shorter in length and lower in off-target rate, and the characteristics enable the CRISPR-Cpf1 to have high application value. However, the use of current gene editing tools for homology directed repair is inefficient and time consuming.

In view of the specificity of the CRISPR-Cpf1 and the CRISPR-Cas9, the construction of a feasible chemical screening system and the identification of a plurality of small molecular compounds for improving the gene editing efficiency are very significant.

It is worth mentioning that the inventor establishes an unbiased drug screening system for high-throughput screening of human pluripotent stem cells, and the unique screening system enables the inventor to discover some novel and interesting small molecule compounds, thereby promoting the CRISPR-Cpf1 mediated precise gene editing process.

The inventor uses OCT4-eGFP-Puro vector to insert gene into hPSCs, then uses puromycin (Puro) to screen cell drug, finally obtains single clone with Puro resistance, and the inventor uses the single clone number as output result to compare the difference between the control group and each experimental group. The inventor screens nearly 600 chemical small molecules in total, and finally discovers that a small molecule compound AZD-7762 can remarkably promote a CRISPR-Cpf1 mediated hPSC gene editing process.

The gene knock-in efficiency can be improved by about 3 times after the small molecule AZD-7762 is added, and the function of the small molecule is obviously superior to that of some chemical small molecules which are found previously. Importantly, the combination of CRISPR-Cpf1 and small molecule compounds provides a simple and efficient strategy for precise gene editing, and can be further developed and applied to gene editing in animals and human germ cells. These advances will undoubtedly provide new methods and tools for genome engineering, accelerating the development of new methods for the treatment of human diseases.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Zhejiang university

<120> small molecule compound promoting gene editing and use thereof

<130> P2017-2500

<160> 89

<170> PatentIn version 3.5

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gagggcctat ttcccatgat tcct 24

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aaaaaaaggt cttctcgaag acccatctac acttagtaga aattcggtgt ttcgtccttt 60

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<212> DNA

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ggtgcctgcc cttctaggaa 20

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<400> 49

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<400> 50

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<400> 51

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<400> 52

aacttcccca tttttggctt g 21

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<400> 54

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<400> 55

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<400> 56

aacttcccca tttttggctt g 21

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<400> 60

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<400> 61

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<400> 62

ggcatggact gtggtcatga g 21

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<400> 63

ggcgtgtcac aagaaggg 18

<210> 64

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<400> 64

ggaggcggag gatgaagt 18

<210> 65

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<400> 65

tgtgggtgac agaatggt 18

<210> 66

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<400> 66

aatgggtcct gcaagtaa 18

<210> 67

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<400> 67

cacgccaaag ccttcata 18

<210> 68

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gaacggatgg gacggtag 18

<210> 69

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<400> 69

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<210> 70

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<400> 70

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<210> 71

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<400> 71

attgaaacgc cacagatt 18

<210> 72

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<400> 72

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<210> 73

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<400> 73

cgtatttcag cgtttgttc 19

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<400> 74

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<210> 75

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<400> 76

gtagccttgg gaaacagc 18

<210> 77

<211> 18

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<400> 77

cataaggtcg ggtcaggg 18

<210> 78

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<400> 78

aagaaagcac caaggcac 18

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<400> 79

acagagtatc gcaggcacaa 20

<210> 80

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<400> 80

cctttccacc cacccttgat 20

<210> 81

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<400> 81

gtgactgttt ccctttccct ct 22

<210> 82

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<400> 82

tgtcatgggg aattgctggt 20

<210> 83

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<400> 83

gaatttaggt gaagggta 18

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<400> 84

acaagtcgag ctgataca 18

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<400> 85

gttgtcagga aatgcagagc c 21

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<400> 86

agtcctacgt ccgagagtga 20

<210> 87

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<400> 87

acatattcaa caggtgccag c 21

<210> 88

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<400> 88

aagtctcatg ggttttgggg g 21

<210> 89

<211> 120

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<213> Intelligent (Homo sapiens)

<400> 89

gcgagatggg gaagatggca gcggccgtgg gctctgtggc gactctggcg actgagccca 60

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Claims (12)

1. Use of a compound, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof, for the preparation of an enhancer or a formulation for enhancing CRISPR-based gene editing; the compound is

2. The use of claim 1, wherein said gene editing comprises CRISPR-Cpf 1-based gene editing, CRISPR-Cas 9-based gene editing.

3. The use of claim 1, wherein the gene editing is directed against a sample selected from the group consisting of: a cell, a tissue, an organ, or a combination thereof.

4. The use of claim 3, wherein said cells comprise: embryonic stem cells, adipose stem cells, hematopoietic stem cells, immune cells.

5. An in vitro non-diagnostic and non-therapeutic method of promoting CRISPR-based intracellular gene editing, comprising:

(a) Performing gene editing on a cell in the presence of a gene editing promoter, thereby promoting gene editing in the cell,

wherein the gene editing accelerator is a compound AZD-7762, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof.

6. The method of claim 5, wherein the in vitro gene editing is performed in an in vitro reaction system.

7. The method of claim 6, wherein the concentration of the gene-editing promoter in the in vitro reaction system is 0-2 μ M.

8. The method of claim 5, wherein in step (a), comprising:

(a1) Introducing into the cell a nucleotide sequence encoding a Cpf1 nuclease, wherein the Cpf1 nuclease is capable of generating a double-strand break in the target DNA, thereby inducing gene editing of the target DNA; and

(a2) Culturing said cell in the presence of said gene-editing promoting agent.

9. A reagent product or combination of reagents comprising:

(i) A first reagent which is a gene editing accelerator, wherein the gene editing accelerator is a compound AZD-7762, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; and

(ii) A second agent that is an agent that effects CRISPR gene editing.

10. Use of the reagent product of claim 9 for the preparation of a kit for performing CRISPR gene editing.

11. The use according to claim 10, wherein the gene is selected from the group consisting of: OCT4, ALBUMIN, ALKBH1 or combinations thereof.

12. A kit, comprising:

(i) The reagent comprises a first container and a first reagent positioned in the first container, wherein the first reagent is a gene editing accelerator which is a compound AZD-7762, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; and

(ii) A second container, and a second agent located within the second container, the second agent being an agent that performs CRISPR gene editing.

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