CN110241135A - Compound Screening and Identification for Facilitating Gene Editing and Their Applications - Google Patents

Abstract

The present invention provides a kind of screening compounds for promoting gene editing and identification and its application.Specifically, the present invention provides the purposes of a kind of compound shown in formula A or its pharmaceutically acceptable salt, they be used to prepare the promotor or preparation for promoting gene editing, and wherein formula A structure is as noted in the discussion.The compounds of this invention is remarkably improved the efficiency of CRISPR gene editing, so that a kind of simple and efficient strategy is provided for accurately gene editing, to provide new Method and kit for for genome project.

Description

Compound Screening and Identification for Facilitating Gene Editing and Their Applications

technical field

The present invention relates to the field of biology, and more particularly relates to the screening and identification of compounds for promoting gene editing and its application.

Background technique

With the emergence of gene editing technology, it is possible to effectively edit and transform different cells. Taking pluripotent stem cells as an example, it includes embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), which can be used to study 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.).

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

With the discovery of nucleases such as Cas9, some gene editing technologies based on CRISPR technology, such as CRISPR-Cas9-mediated gene editing, have been developed.

Some small molecule compounds have been found to regulate the gene editing process mediated by CRISPR-Cas9. 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 improve the efficiency of CRISPR-Cas9-mediated gene editing.

The gene editing technology based on CRISPR-Cpf1 nuclease is another targeted gene editing technology, which expands the scope of gene editing and has higher precision. However, the gene editing efficiency of CRISPR-Cpf1 is still unsatisfactory. Furthermore, chemical small molecules that can facilitate CRISPR-Cpf1 gene editing have not been reported yet.

Therefore, there is an urgent need in this field to develop new compounds that can effectively improve gene editing efficiency.

Contents of the invention

The purpose of the present invention is to provide a compound capable of effectively improving gene editing efficiency and its application.

In the first aspect of the present invention, there is provided a compound represented by formula A, or a pharmaceutically acceptable salt thereof, or an optical isomer or a racemate thereof, or a solvate thereof, for Preparation of accelerators or preparations that promote gene editing;

In the formula,

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;

Each R2 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, -(L2)q-substituted or unsubstituted C1-C6 alkyl;

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

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 ring alkyl;

R4 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 ring alkyl;

R5 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 ring Alkyl, -(L2)q-substituted or unsubstituted C1-C6 alkyl;

R7 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 ring alkyl;

Wherein, p is 0, 1, 2, or 3; and each L1 is independently selected from the following group: -CH ₂ -, -NH-, -S-, -O-, or a combination thereof;

q is 0, 1, 2, or 3; and each L2 is independently selected from the group consisting of -SO ₂ -, -CH ₂ -, -NH-, -S-, -O-, or a combination thereof;

Ra and Rb are each independently selected from the group consisting of H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C5 cycloheteroalkyl, wherein The cycloheteroalkyl group is 5-7 members and contains 1-3 heteroatoms selected from N, O, and S;

In another preferred example, each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb);

In another preferred example, n is 0, 1 or 2;

In another preferred example, each R2 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb), -(L2)q-substituted or unsubstituted C1-C3 alkyl;

In another preferred embodiment, m is 0, 1, or 2;

In another preferred example, R3 is selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb);

In another preferred example, R4 is selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb).

In another preferred example, R5 is selected from the group consisting of H, halogen, -(L1)p-substituted or unsubstituted C1-C6 alkyl, -(L1)p-N(Ra)(Rb); and/or

R6 is selected from the group consisting of H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb), -(L2)q-substituted or unsubstituted C1-C6 alkyl;

In another preferred embodiment, R7 is selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb);

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

In another preferred example, each L1 is independently selected from the following group: -CH ₂ -, -NH-, -O-, or a combination thereof;

In another preferred embodiment, q is 0, 1, or 2;

In another preferred example, each L2 is independently selected from the following group: -SO ₂ -, -NH-, -O-, or a combination thereof;

In another preferred embodiment, Ra and Rb are each independently selected from the following group: H, substituted or unsubstituted C1-C3 alkyl;

In another preferred embodiment, each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl;

In another preferred embodiment, n is 0 or 1;

In another preferred example, each R2 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl, -N(Ra)(Rb), -(L2)q-substituted or unsubstituted C1-C3 alkyl;

In another preferred embodiment, m is 0 or 1;

In another preferred example, R3 is selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl, -N(Ra)(Rb);

In another preferred example, R4 is selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl, -N(Ra)(Rb);

In another preferred example, R5 is selected from the following group: -(L1)p-substituted or unsubstituted C1-C3 alkyl, -(L1)p-N(Ra)(Rb);

In another preferred example, R6 is selected from the following group: substituted or unsubstituted C1-C6 alkyl, -N(Ra)(Rb), -(L2)q-substituted or unsubstituted C1-C6 alkyl;

In another preferred example, R7 is selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl, -N(Ra)(Rb);

In another preferred example, each L1 is independently selected from the following group: -CH ₂ -, -NH-, or a combination thereof;

In another preferred embodiment, each L2 is independently: -SO ₂ -;

In another preferred embodiment, Ra and Rb are each independently selected from the following group: H, unsubstituted C1-C3 alkyl;

In another preferred example, n is 0;

In another preferred embodiment, m is 0;

In another preferred embodiment, R3 is H;

In another preferred embodiment, R4 is H;

In another preferred embodiment, R5 is -CH ₂ -NH-CH ₃ ;

In another preferred embodiment, R6 is -SO ₂ -(C1-C3 alkyl), more preferably -SO ₂ -C ₃ H ₇ ;

In another preferred embodiment, R7 is -N(Ra)(Rb), more preferably -NH ₂ ;

In another preferred example, the compound is

In another preferred example, the gene editing includes CRISPR-based gene editing.

In another preferred example, the gene editing includes gene editing based on CRISPR-Cpf1 and gene editing based on CRISPR-Cas9;

In another preferred example, the gene editing includes in vivo gene editing, in vitro gene editing, or a combination thereof;

In another preferred example, the sample targeted by the gene editing is selected from the group consisting of cells, tissues, organs, or combinations thereof;

In another preferred example, the samples are from animals, plants, microorganisms (including bacteria and viruses);

In another preferred example, the samples are from humans and non-human mammals;

In another preferred example, the cells include primary cells and passaged cells;

In another preferred example, the cells include somatic cells, germ cells, and stem cells;

In another preferred example, the stem cells include: totipotent stem cells, pluripotent stem cells, and multipotent stem cells;

In another preferred embodiment, the stem cells are induced pluripotent stem cells (hiPSC);

In another preferred example, the cells include: embryonic stem cells, fat stem cells, hematopoietic stem cells, immune cells (such as T cells, NK cells);

In another preferred example, the preparation includes a pharmaceutical composition.

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

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

Wherein, the gene editing promoter is a compound represented by formula A, or a pharmaceutically acceptable salt thereof, or an optical isomer or a racemate thereof, or a solvate thereof; as defined in Requirement 1);

In another preferred embodiment, in step (a), before, during and/or after gene editing, the gene editing promoter is contacted with the gene editing cells;

In another preferred example, the in vitro gene editing is performed in an in vitro reaction system;

In another preferred example, in the in vitro reaction system, the concentration of the gene editing accelerator is 0-2 μM;

In another preference, in step (a), including:

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

(a2) cultivating the cells in the presence of the gene editing promoter;

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

In a third aspect of the present invention, a reagent product (or reagent combination), comprising:

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

(ii) a second reagent, the second reagent is a reagent for CRISPR gene editing;

In another preferred example, the second reagent includes one or more reagents selected from the following group:

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

(c2) Cas9 nuclease, the coding sequence of Cas9 nuclease, or the carrier expressing Cas9 nuclease, or combination;

(c3) crRNA, a carrier for producing said crRNA;

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

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

In another preferred example, the kit also contains instructions;

In another preferred example, the description describes the method for promoting gene editing of the present invention;

In another preferred example, the gene editing is gene editing for somatic cells and stem cells;

In another preferred example, the gene editing is CRISPR-Cpf1-based gene editing;

In another preferred embodiment, the cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, and human embryonic kidney 293T cells;

In another preferred example, the gene editing is aimed at pathogenic genes, tumor-related genes (such as oncogenes), immune-related genes (such as genes related to autoimmunity), and vision-related genes;

In another preferred embodiment, the gene is selected from the group consisting of OCT4, ALBUMIN, ALKBH1 or a combination thereof.

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

(i) a first container, and a first reagent located in the first container, the first reagent is a gene editing accelerator, and the gene editing accelerator is a compound shown in formula A, or its pharmaceutical An 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); and

(ii) a second container, and a second reagent located in the second container, where the second reagent is a reagent for CRISPR gene editing.

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

In another preferred example, the subject includes humans and non-human mammals;

In another preferred example, the gene editing reagents include gene editing reagents based on CRISPR-Cpf1.

In another preferred embodiment, before, during and/or after the administration of the gene editing promoter, the subject is administered a gene editing agent for gene editing.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows that knockout hPSC lines were efficiently generated with CRISPR-Cpf1.

Figure 2 shows the identification of small molecules capable of significantly promoting CRISPR-Cpf1-mediated gene knock-in in human pluripotent stem cells.

Figure 3 shows small molecules that can significantly promote the establishment of CRISPR-Cpf1-mediated hPSC knock-in cell lines.

Figure 4 shows the working model of CRISPR-Cpf1

Figure 5 shows the identification model for CRISPR-Cpf1-producing knockout hPSC lines

Figure 6, 7 shows the analysis of potential off-target sites for CRISPR-Cpf1

Figure 8 shows that candidate small molecules can significantly promote CRISPR-Cpf1 or CRISPR-Cas9-mediated hPSC gene knock-in at the OCT4 locus

Figure 9 shows toxicity assays for candidate small molecules

Figure 10 shows that candidate small molecules can significantly promote CRISPR-Cpf1 or CRISPR-Cas9-mediated hPSC gene knock-in at the ALBUMIN site

Figure 11 shows that candidate small molecules can significantly promote CRISPR-Cpf1-mediated hPSC double gene knock-in

Detailed ways

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that a compound with a structure as shown in Formula A can significantly improve the gene editing efficiency of CRISPR, especially the efficiency of gene knock-in mediated by CRISPR-Cpf1 . Experiments have shown that the compound of formula A (such as VE-822) can significantly promote the efficiency of gene editing mediated by CRISPR-Cpf1, for example, high-efficiency gene editing in human pluripotent stem cells to generate gene knock-in cell lines. On this basis, the inventors have completed the present invention.

the term

The term "C ₁ -C ₈ alkylene" refers to a straight or branched chain alkylene having 1 to 8 carbon atoms, such as methylene, ethylene, propylene, butylene, or the like.

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

The term "C ₃ -C ₆ cycloalkyl" refers to a cycloalkyl group having 3-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or similar groups.

The term "C ₁ -C ₃ alkylene" refers to a straight or branched chain alkylene 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 5-7 carbon atoms, such as cyclopentyl, cyclohexyl, or the like.

The term "halogen" refers to F, Cl, Br and I.

gene editing booster

As used herein, "compound of the present invention", "compound of formula A", and "gene editing accelerator of the present invention" are used interchangeably, referring to the compound of the structure shown in formula A, or a pharmaceutically acceptable salt thereof, or An optical isomer or a racemate thereof, or a solvate thereof. It should be understood that the term also includes mixtures of the aforementioned components.

In the formula, the definition of each group is as above.

The compound of the present invention has a very significant promotion effect on the efficiency of gene editing

In the present invention, pharmaceutically acceptable salts of the compound 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 a base which is suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, 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, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.

The compound of formula A of the present invention can be prepared by methods well known to those skilled in the art, and there is no special limitation on the reaction parameters of each step.

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

gene editing

The compound of the present invention can significantly improve the efficiency of gene editing.

In the present invention, representative gene editing includes (but not limited to): CRISPR-based gene editing. Typically, CRISPR-based gene editing includes CRISPR-Cpf1-based gene editing and CRISPR-Cas-based gene editing. Wherein, the Cas includes Cas9 and the like.

A preferred gene editing is CRISPR-Cpf1 gene editing. CRISPR-Cpf1 can specifically recognize the thymidine nucleotide (T)-rich PAM sequence (TTTN), expanding the scope of RNA-mediated gene editing. CRISPR-Cpf1 generates 5nt cohesive ends that initiate different DNA repair processes. The crRNA length of CRISPR-Cpf1 is much shorter than that of Cas9, which is easier to synthesize in vitro and more suitable for multiple gene editing process. CRISPR-Cpf1 also had a lower off-target rate.

use

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

The above-mentioned compound of formula A of the present invention can be used to improve the efficiency of CRISPR-mediated gene editing, and then can be used to prevent or treat diseases related to pathogenic genes.

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

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

Preferably, said subjects include humans and non-human mammals (rodents, rabbits, monkeys, livestock, dogs, cats, etc.).

Composition and method of application

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

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should match the mode of administration.

Taking the pharmaceutical composition as an example, the composition of the present invention can be prepared in the form of injection, for example, by normal methods using physiological saline or aqueous solution containing glucose and other auxiliary agents. 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 can also be made into powder for nebulized inhalation.

For the pharmaceutical composition of the present invention, it can be administered to desired subjects (such as humans and non-human mammals) by conventional means. Representative modes of administration include, but are not limited to: oral administration, injection, topical administration, and the like.

The main advantages of the present invention include:

(a) For the first time, a small molecule compound that can significantly promote gene editing is provided, which is especially effective for CRISPR-Cpf1/Cas9-mediated knock-in editing of hPSC genes.

(b) The present invention provides a simple and efficient strategy for precise gene editing based on the combination of the compound of formula A and CRISPR-Cpf1.

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

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to 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 suggested conditions. Percentages and parts are by weight unless otherwise indicated.

general method

Plasmid construction

For the pCpfcr vector, the BbsI enzyme recognition sequence and DR (direct repeat) sequence were designed in the upstream primer of the U6 promoter; the U6 promoter region was amplified by PCR using the pUcm-T Vector Cloning Kit (Sangon Biotech Company) and then Cloned into T vector. When constructing the pCpfcr-crRNA vector to express the target sequence (crRNA) of Cpf1, first synthesize a 24bp oligonucleotide according to the crRNA sequence, anneal to form a DNA double-stranded fragment, and then clone it into the pCpfcr vector digested with BbsI.

Regarding the construction of the OCT4-tdTomato donor plasmid, the OCT4-2A-mOrange donor plasmid (Addgene, Plasmid #66986) was used to remove the mOrange sequence as the backbone by PCR, and then the tdTomato sequence was amplified by PCR, and the Gibson Assembly kit (New England Biolabs) ligated the tdTomato sequence to the backbone. All vectors were verified by Sanger sequencing.

cell culture

hPSCs were cultured according to conventional methods. H1 embryonic stem cell line, MEL1 embryonic stem cell line and hiPSCs were cultured with hPSC medium consisting of: DMEM/F12 (Life Technologies), 20% serum-free KSR (Life Technologies), 1X non-essential amino acids (Life Technologies), 100X Penicillin/streptomycin (Life Technologies), 0.055 mM mercaptoethanol (Sigma), and 10 ng/mL bFGF (Peprotech).

Every 3-6 days, hPSC cells were digested with Accutase (Life Technologies) and passaged at a ratio of 1:3 to 1:6. Add 0.5 μM Thiazovivin to the hPSC medium every time you passage or thaw the cells to help the stem cells adhere to the wall and survive.

electroporation

Prepare the electroporation after extracting the plasmid with the ZymoPURE Plasmid Extraction Kit. First, take 81.82 μL Solution Ⅰ and 18,18 μL Supplement Ⅰ (Human Stem Cell Kit 1, Lonza) was mixed with the plasmid to prepare the electrotransfer solution.

For gene knock-in experiments, the plasmid consisted of 3 μg pcDNA3.1-hLbCpf1 (Addgene, plasmid number #69988), 3 μg pCpfcr-crRNA and 4 μg of OCT4-tdTomato donor or OCT4-2A-eGFP-PGK-Purodonor (Addgene, plasmid Part #31938) composition. The hPSC cells were digested into single cells with Accutase, and 1×10 ⁶ cells were resuspended in electroporation solution, and electroporated with Amaxa electroporator (Lonza). After electroporation, cells were seeded into one well of a six-well plate and cultured for 2-3 days. Afterwards, the knock-in efficiency was analyzed by flow cytometry or T7E I.

For established cloning experiments, seed 500-2000 cells into 10 cm dishes and culture for 4-7 days. Clones were picked for expansion and storage. After 2 weeks, immunostaining, flow cytometry, sequencing and other methods were used to identify cell lines expressing tdTomato.

transfection

Human embryonic kidney 293T cells were cultured in DMEM (Life Technologies) plus 10% fetal bovine serum, 100×penicillin/streptomycin medium. When the 293T cell density reached 70%-80%, the plasmids were mixed with Lipofectamine ^™ 3000 transfection reagent (Invitrogen) for transfection. The plasmid mix consisted of 1 μg of pY016(pcDNA3.1-hLbCpf1) and 1 μg of pCpfcr-crRNA. 2-3 days after transfection, the T7EI assay was used for analysis.

PFLP and T7E Ⅰ analysis

Cells were collected 2-3 days after transfection or electroporation, and the genome was extracted with the Rapid Genomic DNA Mini-Isolation Kit (ZYMO). Genomic fragments with CRISPR targets were amplified with Taq polymerase (Vazyme).

For RFLP experiments, 2 μL of the amplified product was digested with restriction enzymes and run on a 2% agarose gel for electrophoresis analysis. For point mutations, 100 μL of the PCR product was purified and recovered with a DNA Clean&Concentrator-5 (ZYMO RESEARCH) kit, digested with NcoI enzyme (New England Biolabs) and analyzed by electrophoresis on a 2% agarose gel. The mutation rate was calculated by the formula 100×(b+c)/(a+b+c), where a represents the band intensity of the product not digested by NcoI, and b and c represent the band intensity of the product after digestion.

For the T7E Ⅰ experiment, 10 μL of the amplified product was mixed with NEB buffer 2 (New England Biolabs) to form a system with a total volume of 16 μL. The mixed system was incubated at 95°C for 5 minutes, then lowered to 85°C at a rate of 2°C per second, then slowly lowered to 25°C at a rate of 0.1°C per second, and finally incubated at 4°C. T7EI enzyme (NewEngland Biolabs) was added to 16 μL of the reaction product to form a system with a total volume of 20 μL and incubated at 37° C. for 30 minutes, and then analyzed by electrophoresis on 2.5% agarose gel. Gel images were taken by JS-2000 Gel Imager (Peiqing Science&Technology) and analyzed by ImageJ software.

The mutation rate is calculated by the formula 100×(1–(1–(b+c)/(a+b+c)) ^1/2 ), where a represents the band intensity of the product not digested by T7EⅠ, b and c Respectively represent the product band intensity after enzyme digestion.

Flow Cytometry

hPSCs transfected with Cpf1, OCT4-crRNA, and OCT4-tdTomato vectors were seeded in one well of a six-well plate and cultured with hPSC medium for 3–4 days. Differentiated and undifferentiated cells were digested with Accutase for 3-5 minutes into single cells. Then resuspend with 0.3-1mL 1X phosphate buffered saline. Finally, the data was obtained by Beckman flow cytometer, and the results were analyzed.

hPSC differentiation

About 200 undifferentiated OCT4-tdTomato hPSC cells were seeded in one well of a six-well plate and cultured in hPSC medium for 4-7 days. Then change to differentiation medium (DMEM/F12, 20% serum replacement, 1 × NEAA, 100 × penicillin/streptomycin (Life Technologies), 0.055mM 2-mercaptoethanol (Sigma), 0.1 μM LDN225, 10 μM E616452) culture 3 days. After the fourth or fifth day, cells can be analyzed by flow cytometry and immunostaining.

Immunostaining

Cells were fixed with 4% paraformaldehyde for 10-15 minutes at room temperature, and then washed three times with 1x PBST (1x PBS+0.3% TritonX-100 (Vetec)) for 5 minutes each. Next, cells were incubated in blocking solution (1x PBST+5% BSA (fetal bovine serum)) at room temperature for 0.5-1 hour, and the primary antibody was incubated overnight (4°C). Subsequently, wash three times with PBST at room temperature, each time for 15 minutes, then add the corresponding secondary antibody and incubate for 1 hour. Finally, nuclei were stained using Hoechst (1:5000).

Apoptosis analysis

Apoptosis was analyzed by the Apoptosis Kit. Human pluripotent stem cells were first treated with DMSO, AZD-7762 (1 μM) and VE-822 (1 μM). Collect ^5x105 cells and wash twice with ice-cold PBS. Cells were centrifuged at 4°C for 5 minutes, then resuspended in 50 μL 1x Binding Buffer, added 2.5 μL Annexin V(AV)-FITC and 2.5 μL PI staining solution and incubated at room temperature for 10-15 minutes. Then add 250μL of 1x Binding Buffer to the mixture. Finally, cell apoptosis was detected by flow cytometry.

Off-target analysis

Cas-OFFinder can be used to detect potential off-target sites. The set mismatch value is less than or equal to 6. After finding potential off-target sites, primers were designed to amplify the off-target site fragments on the cell genome by PCR, and then sequenced and identified by Sanger sequencing.

Statistical analysis

P values were obtained by two-tailed t-test, and all statistical values were expressed as mean±s.e.

Example 1.

Efficient construction of 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 driven by the U6 promoter was constructed (Figures 4a and 4b).

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

To construct gene-specific crRNA plasmids, a panel of crRNAs specifically targeting these genes was designed (Fig. 1b and Table 1).

【Table 1】

Construction of nucleic acid sequences expressing crRNA plasmids

PCR primers for constructing plasmids

PCR primers for genotyping and sequencing

PCR primers for T7EI experiments

Primers for RFLP experiments

Primers for qPCR

PCR primers for off-target analysis

ssODN template sequence

In 293T cells, the genome editing ability of these crRNAs was tested. In the T7E1 (T7 endonuclease I) assay, a 20-30% indel ratio was observed (Fig. 4c).

Then, the ability of CRISPER/Cpf1-mediated genome editing in hPSCs was investigated (Fig. 1a). In the T7E1 assay, ALKBH1 and CLEC16A knockout efficiency was also observed to be 20–30% in hESCs and hiPSCs (Fig. 1c).

To further establish knockout hPSC lines, transfected hPSCs were passaged at low cell density, clones were picked, and analyzed by PCR genotyping (Fig. 1a).

For ALKBH1, 20.8% of the clones had a monoallelic knockout and 12.5% had a biallelic knockout (Fig. 1e).

For CLEC16A, 25% of the clones were mono-allelic and 16.7% of the clones were bi-allelic (Fig. 1e).

Sanger sequencing results confirmed that after gene knockout occurred at the ALKBH1 and CLEC16A target sites, the genome sequence lacked some bases, thus achieving gene knockout (Fig. 1f).

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

Altogether, the above experimental results clearly demonstrate that CRISPR-Cpf1 can efficiently perform gene knockdown in hPSCs.

Example 2.

Screening of small molecules that can significantly promote CRISPR-Cpf1-mediated HDR in hPSCs

In this example, in order to test the ability of CRISPR-Cpf1 for gene insertion, three plasmids were transferred into hPSCs by electroporation: one plasmid expresses Cpf1, one contains a specific crRNA targeting OCT4, and contains the eGFP reporter gene and puromycin (Puro) resistant HDR template plasmid (Fig. 8a). After electroporation, hPSCs were first cultured with ordinary medium for two days, and then puromycin was added to the medium for 3-4 days. It was observed that the efficiency of genome repair by HDR approach is relatively low and needs to be further improved.

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

result:

Experiments showed that SCR7, a small molecule known to promote CRISPR-Cas9-mediated gene knock-in, had no significant promoting effect in CRISPR-Cpf1-mediated gene knock-in (Fig. 2e).

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

Example 3

Chemical Small Molecule Screening

In this example, hundreds of candidate compounds were screened and tested one by one.

In this example, to improve the efficiency of CRISPR-Cpf1-mediated gene editing in hPSCs, large-scale compound screening was performed using the OCT4-eGFP knock-in selection system with puromycin (Fig. 2a).

Specifically, three plasmids were transferred into hPSCs by electroporation: one expressing Cpf1, one containing a specific crRNA targeting OCT4, and an HDR template plasmid containing an eGFP reporter gene and puromycin (Puro) resistance ( Figure 8a).

For each candidate compound, 100 μL of cell culture medium and 0.2 μL of screened small molecules (1:1000) were added to each well of a 48-well plate before electroporation. After cell electroporation, ^1x106 cells were mixed in 4.8mL hPSC medium supplemented with Thiazovivin small molecule and seeded in a 48-well plate. The cells were cultured in the medium supplemented with VE-822 small molecule, and after two days, the addition of VE-822 small molecule was stopped, and 1 μg/mL puromycin was added to the medium to continue culturing for 3-5 days. Afterwards, the selected clones with puromycin resistance were counted to test the efficiency of the small molecule.

result:

A total of about 600 small molecules in the kinase library were screened, among which the small molecule VE-822 significantly increased the number of hPSC clones (Fig. Compound VE-822 can significantly improve gene editing efficiency.

Example 4

Validation of the effect of gene editing promoters

In this example, further experiments were conducted to verify the effect of VE-822. Test method is the same as embodiment 3.

The results showed that VE-822 reached its maximum effect at 1 μM (Fig. 2d). VE-822 is an inhibitor of ATR kinase, which can promote gene knock-in efficiency by 5.9 times.

In addition, VE-822 did not significantly increase the cell proliferation rate of hPSCs, indicating that VE-822 did not increase the gene knock-in efficiency by promoting cell proliferation (Fig. 9a).

Furthermore, VE-822 did not exhibit cytotoxicity against hPSCs (Figures 9c and 9d). hPSCs treated with VE-822 still highly expressed specific pluripotency genes such as OCT4 and NANOG (Fig. 9b).

In contrast, in the case of CRISPR-Cpf1-mediated knock-in in hPSCs, the control compound SCR7 had no significant promotion effect, but VE-822 could significantly promote Cpf1-mediated gene knock-in in hPSCs (Fig. 2e and 8d).

In the CRISPR-Cas9 system, the efficiency of SCR7 was less than 2-fold, while the effect of VE-822 was more significant (about 4-fold).

In addition, knockdown of ATR gene with siRNAs could promote CRISPR-Cpf1-mediated gene knock-in efficiency, indicating that VE-822 indeed works by targeting ATR (Fig. 2g). The above results successfully confirmed that VE-822 can significantly improve the efficiency of CRISPR-Cpf1-mediated gene knock-in in hPSCs.

Example 5.

Small molecules significantly promote CRISPR-Cpf1-mediated gene knock-in cell line generation in hPSCs

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

The method was as follows: Three plasmids were electroporated into hPSCs: one expressing Cpf1, one containing crRNA specifically targeting OCT4, and one donor plasmid containing OCT4-2A-tdTomato (Fig. 3a). After 5-6 days, tdTomato-positive cells were observed, and the efficiency of gene knock-in was detected by FACS (flow cytometry) analysis.

The results showed that VE-822 could significantly increase the percentage of tdTomato-positive cells, which indicated that VE-822 could promote the gene knock-in of CRISPR-Cpf1 in hPSCs (Fig. 3b, c).

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

After differentiation of these stem cells, immunostaining and FACS experiments could prove 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 gene (Fig. 3d, e). Therefore, OCT4-tdTomato can simultaneously reflect the expression of the endogenous pluripotency gene OCT4 during hPSCs stage and differentiation.

Afterwards, sequencing was performed using the Sanger method. The sequencing results suggested 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 of OCT4 and ALBUMIN proved that VE-822 can significantly promote the efficiency of CRISPR-Cpf1-mediated double gene knock-in (Fig. 11a).

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

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

discuss

In the present invention, the present invention proves through experiments that gene editing based on CRISPR-Cpf1 can perform efficient gene knockout and insertion editing in human pluripotent stem cell lines. The results of the T7EI experiment proved that CRISPR-Cpf1 successfully cut the genome at specific sites of the two genes of ALKBH1 and CLEC16A. Afterwards, genotype identification was carried out by picking a single clone, and the results showed that the knockout efficiency of this gene was relatively high.

From the results of sequencing, it can also be concluded that CRISPR-Cpf1 can successfully construct gene knockout cell lines.

For the gene insertion experiment, the inventor constructed the OCT4-tdTomato vector, and successfully inserted a red fluorescent reporter gene into the OCT4 gene through CRISPR-Cpf1-mediated gene editing technology, and it can be detected by flow cytometry and immunofluorescence staining experiments into hPSCs successfully inserted with a fluorescent reporter gene.

In addition, CRISPR-Cpf1 is unique in that it has a shorter crRNA length and a lower off-target rate, which make it of high application value. However, using current gene editing tools to perform homology-directed repair is inefficient and time-consuming.

In view of the specificity of CRISPR-Cpf1 and CRISPR-Cas9, it is very meaningful to construct a feasible chemical screening system and identify some small molecular compounds that can improve gene editing efficiency.

It is worth mentioning that the inventors established an unbiased drug screening system for high-throughput screening of human pluripotent stem cells, and this unique screening system enabled the inventors to discover some novel and interesting small molecules Compounds, thereby promoting the precise gene editing process mediated by CRISPR-Cpf1.

The inventors used the OCT4-eGFP-Puro vector to insert genes into hPSCs, and then used puromycin (Puro) to screen the cells for drugs, and finally obtained a single clone with Puro resistance. The inventors calculated the number of single clones As the output result, compare the difference between the control group and each experimental group. The inventors screened a total of nearly 600 small chemical molecules, and finally found that the small molecule compound VE-822 can significantly promote the gene editing process of hPSC mediated by CRISPR-Cpf1.

After adding the small molecule VE-822, the gene knock-in efficiency can be increased by 5.9 times, and the function of this small molecule is significantly better than some previously discovered chemical small molecules. Importantly, the combination of CRISPR-Cpf1 and small molecule compounds provides a simple and efficient strategy for precise gene editing, which can also be further developed and applied to gene editing in animals and human germ cells. Undoubtedly, these advances will provide new methods and tools for genome engineering and accelerate the development of new treatments for human diseases.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Zhejiang University

<120> Compound screening and identification for gene editing and its application

<130> P2017-2499

<160> 89

<170> PatentIn version 3.5

<210> 1

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 1

tttccccctg tctccgtcac cactctgg 28

<210> 2

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 2

agatcccctg tctctgtcac cactctgg 28

<210> 3

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 3

aaaaccagag tggtgacaga gacagggg 28

<210> 4

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 4

ccctgtctcc gtcaccactc tgg 23

<210> 5

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 5

caccgccctg tctccgtcac cactc 25

<210> 6

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 6

aaacgagtgg tgacggagac agggc 25

<210> 7

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 7

tgagcccggg gaggacgcct ttcggaaa 28

<210> 8

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 8

agatcgaaag gcgtcctccc cgggctca 28

<210> 9

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 9

aaaatgagcc cggggaggac gcctttcg 28

<210> 10

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 10

tttgccccgc gtcttggagg gacttacg 28

<210> 11

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 11

agatccccgc gtcttggagg gacttacg 28

<210> 12

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 12

aaaacgtaag tccctccaag acgcgggg 28

<210> 13

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 13

cccactaacc gtcacactta gcaataaa 28

<210> 14

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 14

agatttgcta agtgtgacgg ttagtggg 28

<210> 15

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 15

aaaacccact aaccgtcaca cttagcaa 28

<210> 16

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 16

tttcacctcc agaccgattg ttattgaa 28

<210> 17

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 17

agatacctcc agaccgattg ttattgaa 28

<210> 18

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 18

aaaattcaat aacaatcggt ctggaggt 28

<210> 19

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 19

tttcggaaac ttttccgctt ctaccgtc 28

<210> 20

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 20

agatggaaac ttttccgctt ctaccgtc 28

<210> 21

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 21

aaaagacggt agaagcggaa aagtttcc 28

<210> 22

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 22

tttgaccaaa aacaccacag tcacagaa 28

<210> 23

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 23

agataccaaa aacaccacag tcacagaa 28

<210> 24

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 24

aaaattctgt gactgtggtg tttttggt 28

<210> 25

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 25

gccttaggct tataacatca catttaaa 28

<210> 26

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 26

agataatgtg atgttataag cctaaggc 28

<210> 27

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 27

aaaagcctta ggcttataac atcacatt 28

<210> 28

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 28

ccttaggctt ataacatcac att 23

<210> 29

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 29

caccgaatgt gatgttataa gccta 25

<210> 30

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 30

aaactaggct tataacatca cattc 25

<210> 31

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 31

gagggcctat ttcccatgat tcct 24

<210> 32

<211> 67

<212>DNA

<213> Artificial Sequence

<400> 32

aaaaaaaggt cttctcgaag acccatctac acttagtaga aattcggtgt ttcgtccttt 60

ccacaag 67

<210> 33

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 33

ggtgcctgcc cttctaggaa 20

<210> 34

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 34

aggaccgggg ttttcttcca 20

<210> 35

<211> 40

<212>DNA

<213> Artificial Sequence

<400> 35

tggaagaaaa ccccggtcct atggtgagca agggcgagga 40

<210> 36

<211> 40

<212>DNA

<213> Artificial Sequence

<400> 36

ttcctagaag ggcaggcacc ttacttgtac agctcgtcca 40

<210> 37

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 37

atcttcagga ggtaagggtg 20

<210> 38

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 38

cgatctcgaa ctcgtggc 18

<210> 39

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 39

gcttccatca ctggctcgta 20

<210> 40

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 40

cgacatcccc tgcttgtttc 20

<210> 41

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 41

aaattccact actcccacat ctcc 24

<210> 42

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 42

cggattccca ggctcttttg 20

<210> 43

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 43

acccttcaaa gcattgtctg c 21

<210> 44

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 44

aacttcccca tttttggctt g 21

<210> 45

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 45

atgtccgtga gcttccgtc 19

<210> 46

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 46

cgatctcgaa ctcgtggc 18

<210> 47

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 47

gatcaagcag cgactatgca 20

<210> 48

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 48

tcacttgggt atgagcattg 20

<210> 49

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 49

aaattccact actcccacat ctcc 24

<210> 50

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 50

cggattccca ggctcttttg 20

<210> 51

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 51

acccttcaaa gcattgtctg c 21

<210> 52

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 52

aacttcccca tttttggctt g 21

<210> 53

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 53

aaattccact actcccacat ctcc 24

<210> 54

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 54

cggattccca ggctcttttg 20

<210> 55

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 55

acccttcaaa gcattgtctg c 21

<210> 56

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 56

aacttcccca tttttggctt g 21

<210> 57

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 57

atatgaagcg tgccgtagac t 21

<210> 58

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 58

tgcctatgtc tggctctatt ctg 23

<210> 59

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 59

ggccaaaggc agttgtattg a 21

<210> 60

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 60

gtgagtaccc caaaaatagc agg 23

<210> 61

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 61

tgcaccacca actgcttagc 20

<210> 62

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 62

ggcatggact gtggtcatga g 21

<210> 63

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 63

ggcgtgtcac aagaaggg 18

<210> 64

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 64

ggaggcggag gatgaagt 18

<210> 65

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 65

tgtgggtgac agaatggt 18

<210> 66

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 66

aatgggtcct gcaagtaa 18

<210> 67

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 67

cacgccaaag ccttcata 18

<210> 68

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 68

gaacggatgg gacggtag 18

<210> 69

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 69

ttaggcagac ctctgtga 18

<210> 70

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 70

tgggcaacaa gagcaaag 18

<210> 71

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 71

attgaaacgc cacagatt 18

<210> 72

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 72

ctagccctta gccttgac 18

<210> 73

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 73

cgtatttcag cgtttgttc 19

<210> 74

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 74

gctaagcgag atttcctaa 19

<210> 75

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 75

ccctaaatgg agcgagaa 18

<210> 76

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 76

gtagccttgg gaaacagc 18

<210> 77

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 77

cataaggtcgggtcaggg 18

<210> 78

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 78

aagaaagcac caaggcac 18

<210> 79

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 79

acagagtatc gcaggcacaa 20

<210> 80

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 80

cctttccacc cacccttgat 20

<210> 81

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 81

gtgactgttt ccctttccct ct 22

<210> 82

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 82

tgtcatgggg aattgctggt 20

<210> 83

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 83

gaatttaggt gaagggta 18

<210> 84

<211> 18

<212>DNA

<213> Artificial Sequence

<400> 84

acaagtcgag ctgataca 18

<210> 85

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 85

gttgtcagga aatgcagagc c 21

<210> 86

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 86

agtcctacgtccgagagtga 20

<210> 87

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 87

acatattcaa caggtgccag c 21

<210> 88

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 88

aagtctcatg ggttttgggg g 21

<210> 89

<211> 120

<212>DNA

<213> Homo sapiens

<400> 89

gcgagatggg gaagatggca gcggccgtgg gctctgtggc gactctggcg actgagccca 60

tggaggacgc ctttcggaaa cttttccgct tctaccgtca gagccggccc gggaccgcag 120

Claims (10)

1. a kind of compound shown in formula A or its pharmaceutically acceptable salt or its optical isomer or its racemic modification or The purposes of its solvate, which is characterized in that be used to prepare the promotor or preparation for promoting gene editing；

In formula,

Each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 naphthenic base；

N is 0,1,2,3 or 4；

Each R2 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C3-C6 the naphthenic base ,-substituted or unsubstituted C1-C6 alkyl of (L2) q-；

M is 0,1,2,3 or 4；

R3 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 Alkenyl, substituted or unsubstituted C3-C6 naphthenic base；

R4 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 Alkenyl, substituted or unsubstituted C3-C6 naphthenic base；

R5 is selected from the group: H, the halogen ,-substituted or unsubstituted C1-C6 alkyl of (L1) p- ,-(L1) p-N (Ra) (Rb) ,-(L1) p Substituted or unsubstituted C2-C6 the alkenyl ,-substituted or unsubstituted C3-C6 naphthenic base of (L1) p-；

R6 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 Alkenyl, substituted or unsubstituted C3-C6 the naphthenic base ,-substituted or unsubstituted C1-C6 alkyl of (L2) q-；

R7 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), substituted or unsubstituted C2-C6 Alkenyl, substituted or unsubstituted C3-C6 naphthenic base；

Wherein, 0,1,2 or 3 p；And each L1 is independently selected from the following group :-CH₂,-NH- ,-S- ,-O-, or combinations thereof；

Q is 0,1,2 or 3；And each L2 is independently selected from the following group :-SO₂-、-CH₂,-NH- ,-S- ,-O-, or combinations thereof；

Ra and Rb are each independently selected from the following group: H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C3-C6 ring Alkyl, substituted or unsubstituted C2-C5 cycloheteroalkyl, wherein the cycloheteroalkyl be 5-7 member and containing 1-3 selected from N, O, With the hetero atom of S；

In another preferred example, each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb)；

In another preferred example, 0,1 or 2 n；

In another preferred example, each R2 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb) ,-(L2) the substituted or unsubstituted C1-C3 alkyl of q-；

In another preferred example, 0,1 or 2 m；

In another preferred example, R3 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb)；

In another preferred example, R4 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb).

2. purposes as described in claim 1, which is characterized in that R5 is selected from the group: H, halogen ,-(L1) p- are substituted or unsubstituted C1-C6 alkyl ,-(L1) p-N (Ra) (Rb)；And/or

R6 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb) ,-(L2) q- are substituted or unsubstituted C1-C6 alkyl；

In another preferred example, R7 is selected from the group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb)；

In another preferred example, 0,1 or 2 p；

In another preferred example, each L1 is independently selected from the following group :-CH₂,-NH- ,-O-, or combinations thereof；

In another preferred example, 0,1 or 2 q；

In another preferred example, each L2 is independently selected from the following group :-SO₂,-NH- ,-O-, or combinations thereof；

In another preferred example, Ra and Rb are each independently selected from the following group: H, substituted or unsubstituted C1-C3 alkyl；

In another preferred example, each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl；

In another preferred example, n is 0 or 1；

In another preferred example, each R2 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C3 alkyl ,-N (Ra) (Rb) ,-(L2) the substituted or unsubstituted C1-C3 alkyl of q-；

In another preferred example, m is 0 or 1；

In another preferred example, R3 is selected from the group: H, halogen, substituted or unsubstituted C1-C3 alkyl ,-N (Ra) (Rb)；

In another preferred example, R4 is selected from the group: H, halogen, substituted or unsubstituted C1-C3 alkyl ,-N (Ra) (Rb)；

In another preferred example, R5 is selected from the group: the substituted or unsubstituted C1-C3 alkyl of-(L1) p- ,-(L1) p-N (Ra) (Rb)；

In another preferred example, R6 is selected from the group: substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb) ,-(L2) q- replace Or unsubstituted C1-C6 alkyl；

In another preferred example, R7 is selected from the group: H, halogen, substituted or unsubstituted C1-C3 alkyl ,-N (Ra) (Rb)；

In another preferred example, each L1 is independently selected from the following group :-CH₂,-NH-, or combinations thereof；

In another preferred example, each L2 independently is :-SO₂-；

In another preferred example, Ra and Rb are each independently selected from the following group: H, unsubstituted C1-C3 alkyl；

In another preferred example, 0 n；

In another preferred example, 0 m；

In another preferred example, R3 H；

In another preferred example, R4 H；

In another preferred example, R5 is-CH₂-NH-CH₃；

In another preferred example, R6 is-SO₂(C1-C3 alkyl), is more preferably-SO₂-C₃H₇；

In another preferred example, R7 is-N (Ra) (Rb), is more preferably-NH₂；

In another preferred example, the compound is

3. purposes as described in claim 1, which is characterized in that the gene editing includes that the gene based on CRISPR is compiled Volume.

4. purposes as described in claim 1, which is characterized in that the gene editing includes the base based on CRISPR-Cpf1 Because of editor, the gene editing based on CRISPR-Cas9；

In another preferred example, the gene editing include internal gene editing, outer-gene editor, or combinations thereof；

In another preferred example, the sample that the gene editing is directed to is selected from the group: cell, tissue, organ, or combinations thereof；

In another preferred example, the sample comes from animal, plant, microorganism (including bacterium, virus)；

In another preferred example, the sample comes from people and non-human mammal；

In another preferred example, the cell includes the cell of primary cell and passage；

In another preferred example, the cell includes body cell, reproduction cell, stem cell；

In another preferred example, the stem cell includes: myeloid-lymphoid stem cell, multipotential stem cell, He Zhuanneng stem cell；

In another preferred example, the stem cell is the multipotential stem cell (hiPSC) of induction；

In another preferred example, the cell includes: embryonic stem cell, fat stem cell, candidate stem cell, immunocyte (such as T cell, NK cell)；

In another preferred example, the preparation includes pharmaceutical composition.

5. a kind of external method for promoting genes within cells editor, which is characterized in that the described method includes:

(a) in the presence of gene editing promotor, gene editing is carried out to cell, so that the intracellular gene be promoted to compile Volume,

Wherein, the gene editing promotor is compound shown in formula A or its pharmaceutically acceptable salt or its optics Isomers or its racemic modification or its solvate；(wherein formula A is as defined in claim 1)；

In another preferred example, in step (a), before, during and/or after carrying out gene editing, the gene is compiled Promotor is collected to be contacted with the cell for carrying out gene editing；

In another preferred example, the external gene editing carries out in an external reaction system；

In another preferred example, in the vitro reactions system, the concentration of the gene editing promotor is 0-2 μM；

In another preferred example, in step (a), comprising:

(a1) nucleotide sequence of coding Cpf1 nuclease is imported in Xiang Suoshu cell, wherein the Cpf1 nuclease can be Double-strand break is generated in target DNA, to induce the gene editing of target DNA；With

(a2) in the presence of the gene editing promotor, the cell is cultivated；

In another preferred example, the method is nondiagnostic and non-therapeutic.

6. a kind of reagent manufacture (or reagent combination) characterized by comprising

(i) the first reagent, first reagent are gene editing promotor, and the gene editing promotor is shown in formula A Compound or its pharmaceutically acceptable salt or its optical isomer or its racemic modification or its solvate；(wherein formula A is as defined in claim 1)；With

(ii) the second reagent, second reagent are the reagents for carrying out CRISPR gene editing；

In another preferred example, second reagent includes one or more reagents selected from the group below:

(c1) Cpf1 nuclease, the carrier of the coded sequence of Cpf1 nuclease or expression Cpf1 nuclease or combination；

(c2) Cas9 nuclease, the carrier of the coded sequence of Cas9 nuclease or expression Cas9 nuclease or combination；

(c3) crRNA, the carrier for generating the crRNA；

(c4) for the template with source orientation reparation: single stranded nucleotide sequence or plasmid vector.

7. the purposes of reagent manufacture as claimed in claim 6, which is characterized in that be used to prepare the reagent for carrying out gene editing Box；

In another preferred example, the kit also contains specification；

In another preferred example, the method that the specification describes promotion gene editing of the invention；

In another preferred example, the gene editing is the gene editing for body cell, stem cell；

In another preferred example, the gene editing is the gene editing based on CRISPR-Cpf1；

In another preferred example, the cell is selected from the group: embryonic stem cell induces multi-potent stem cell, human embryo kidney (HEK) 293T cell；

In another preferred example, the gene editing is for Disease-causing gene, tumor-related gene (such as oncogene), immune phase Correlation gene (gene such as relevant to autoimmunity), visual correlation gene；

In another preferred example, the gene is selected from the group: OCT4, ALBUMIN, ALKBH1 or combinations thereof object.

8. a kind of kit characterized by comprising

(i) the first container, and the first reagent in the first container, first reagent are gene editing promotion Agent, the gene editing promotor are compound shown in formula A or its pharmaceutically acceptable salt or its optical isomer Or its racemic modification or its solvate；(wherein formula A is as defined in claim 1)；With

(ii) second container, and the second reagent in the second container, second reagent are to carry out CRISPR The reagent of gene editing.

9. a kind of method for promoting gene editing, which is characterized in that promote comprising steps of applying a gene editing to the object needed Into the gene editing reagent of agent and progress gene editing, the gene editing promotor is compound shown in formula A or its medicine Acceptable salt or its optical isomer or its racemic modification or its solvate on；(wherein in formula A such as claim 1 It is defined)；

In another preferred example, the object includes people and non-human mammal；

In another preferred example, the gene editing reagent includes the gene editing reagent based on CRISPR-Cpf1.

10. method as claimed in claim 9 gives institute before, during and/or after applying the gene editing promotor State the gene editing reagent that object application carries out gene editing.