CN110241135A

CN110241135A - Promote the screening compound of gene editing and identifies and its apply

Info

Publication number: CN110241135A
Application number: CN201810194740.1A
Authority: CN
Inventors: 祝赛勇; 马晓洁
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-03-09
Filing date: 2018-03-09
Publication date: 2019-09-17
Anticipated expiration: 2038-03-09
Also published as: CN110241135B

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

Promote the screening compound of gene editing and identifies and its apply

Technical field

The present invention relates to field of biology, relates more specifically to the screening compound for promoting gene editing and identification and its answer With.

Background technique

With the appearance of gene editing technology, efficient gene editor is carried out for different cells and be transformed into can Energy.By taking multipotential stem cell as an example, it includes embryonic stem cell (hESCs) and induces multi-potent stem cell (hiPSCs), can be used for grinding Study carefully early development and disease development.Therefore, to cell (including body cell, multipotential stem cell etc.) carry out quickly, efficiently, can The gene editing of control is vital.

The nuclease of locus specificity identification can cause DNA double chain break in genome specific position, and in triggering The DNA repair mechanism of source property.In such a way that nonhomologous end engages approach DNA plerosis double-strand break (NHEJ), it will lead to small Segment insertion or missing, the repair mode can be used for generating knockout mutations body.It can then be used to construct with source orientation reparation (HDR) Knock in mutant or reporter cell lines.However, even if being repaired under the assistance of these site specific nucleic acid enzymes by same source orientation Accurate genome editor is carried out again to be still challenging.

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

The gene editing process that the adjustable CRISPR-Cas9 of some small molecule compounds having found is mediated.Yu et al. It was found that the same source orientation reparation that the two small molecules of L755507 and Brefeldin A can promote CRISPR-Cas9 to mediate. Chu and Maruyama et al. have found that the gene editing of CRISPR-Cas9 mediation can be improved in the inhibitor SCR7 of ligase IV Efficiency.

Gene editing technology based on CRISPR-Cpf1 nuclease is another directed gene editing technique, expands base Because of the range of editor and with higher precision, however the gene editing efficiency of CRISPR-Cpf1 is also unsatisfactory.This Outside, the chemical small molecule of CRISPR-Cpf1 gene editing can be promoted to be not yet reported that.

Therefore, there is an urgent need in the art to develop the new compound that can effectively improve gene editing efficiency.

Summary of the invention

The object of the present invention is to provide compound and its applications that one kind can effectively improve gene editing efficiency.

In the first aspect of the present invention, provide a kind of compound shown in formula A or its pharmaceutically acceptable salt or The purposes of its optical isomer or its racemic modification or its solvate is used to prepare the promotor or system for promoting gene editing Agent；

In formula,

Each R1 is independently selected from the following group: H, halogen, substituted or unsubstituted C1-C6 alkyl ,-N (Ra) (Rb), replace or not Substituted 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), replace or not Substituted 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, halogen ,-substituted or unsubstituted C1-C6 the alkyl of (L1) p- ,-(L1) p-N (Ra) (Rb) ,- (L1) the substituted or unsubstituted C2-C6 alkenyl of the p ,-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 naphthenic base, substituted or unsubstituted C2-C5 cycloheteroalkyl, wherein the cycloheteroalkyl is 5-7 member and is selected from containing 1-3 N, the hetero atom of 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, 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) ,-substituted or unsubstituted C1-C3 alkyl of (L2) 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).

In another preferred example, R5 is selected from the group: H, halogen ,-substituted or unsubstituted C1-C6 the alkyl of (L1) p- ,-(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- replace or not Substituted 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) ,-substituted or unsubstituted C1-C3 alkyl of (L2) 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- Substituted 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

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

In another preferred example, the gene editing includes gene editing based on CRISPR-Cpf1, is based on The gene editing of 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 its group It closes；

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, is immunized carefully Born of the same parents' (such as T cell, NK cell)；

In another preferred example, the preparation includes pharmaceutical composition.

In the second aspect of the present invention, a kind of external method for promoting genes within cells editor, the method packet are provided It includes:

(a) in the presence of gene editing promotor, gene editing is carried out to cell, to promote the intracellular gene Editor,

Wherein, the gene editing promotor be 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)；

In another preferred example, in step (a), before, during and/or after carrying out gene editing, by the base Because editor's promotor is 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 energy It is enough that 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.

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

(i) the first reagent, first reagent are gene editing promotor, and the gene editing promotor is formula A Compound represented or its pharmaceutically acceptable salt or its optical isomer or its racemic modification or its solvate；(its Chinese style 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.

In the fourth aspect of the present invention, a kind of kit for being used to prepare and carrying out gene editing is provided；

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 directed to Disease-causing gene, tumor-related gene (such as oncogene), exempts from Epidemic disease related 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.

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

(i) the first container, and the first reagent in the first container, first reagent are gene editing Promotor, the gene editing promotor are that compound shown in formula A or its pharmaceutically acceptable salt or its optics are different Structure body 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 The reagent of CRISPR gene editing.

In the sixth aspect of the present invention, a kind of method for promoting gene editing is provided, comprising steps of to the object needed It applies a gene editing promotor and carries out the gene editing reagent of gene editing, the gene editing promotor is formula A institute The compound shown 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)；

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.

In another preferred example, before, during and/or after applying the gene editing promotor, the object is given Application carries out the gene editing reagent of gene editing.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and in below (eg embodiment) specifically It can be combined with each other between each technical characteristic of description, to form a new or preferred technical solution.As space is limited, herein No longer tire out one by one and states.

Detailed description of the invention

Fig. 1 shows the hPSC cell line that gene knockout is effectively produced with CRISPR-Cpf1.

Fig. 2 shows small point of the gene knock-in in the human pluripotent stem cells that can remarkably promote CRISPR-Cpf1 mediation The identification of son.

Fig. 3 shows building for the small molecule for the hPSC gene knock-in cell line that can remarkably promote CRISPR-Cpf1 mediation It is vertical.

Fig. 4 shows the working model of CRISPR-Cpf1

Fig. 5 shows the identification model that gene knockout hPSC cell line is generated to CRISPR-Cpf1

Fig. 6,7 show the potential Locus Analysis in Shoots that misses the target to CRISPR-Cpf1

Fig. 8 shows that candidate small molecule can remarkably promote CRISPR-Cpf1 or CRISPR-Cas9 in the site OCT4 and mediate HPSC gene knock-in

Fig. 9 shows the toxicity detection to candidate small molecule

Figure 10 shows that candidate small molecule can remarkably promote CRISPR-Cpf1 or CRISPR-Cas9 in the site ALBUMIN The hPSC gene knock-in of mediation

Figure 11 shows that candidate small molecule can remarkably promote the hPSC of CRISPR-Cpf1 mediation and dual-gene knock in

Specific embodiment

The present inventor after extensive and in-depth study, has unexpectedly discovered class formation compound as shown in formula A for the first time The gene editing efficiency that CRISPR can be improved significantly especially can significantly promote the gene knock-in of CRISPR-Cpf1 mediation Efficiency.Experiment shows that the formula A compound (such as VE-822) can significantly promote the gene editing of CRISPR-Cpf1 mediation Efficiency, such as efficient gene editing is carried out in human pluripotent stem cells to generate gene knock-in cell line.On this basis, it sends out Bright people completes the present invention.

Term

Term " C₁-C₈Alkylidene " refers to the linear chain or branched chain alkylidene with 1-8 carbon atom, such as methylene, sub- second Base, propylidene, butylidene or similar group.

Term " C₁-C₆Alkyl " refers to the linear or branched alkyl group with 1-6 carbon atom, such as methyl, ethyl, propyl, different Propyl, butyl, isobutyl group or similar group.

Term " C₃-C₆Naphthenic base " refers to the naphthenic base with 3-6 carbon atom, for example, cyclopropyl, cyclobutyl, cyclopenta or Similar group.

Term " C₁-C₃Alkylidene " refers to the linear chain or branched chain alkylidene with 1-3 carbon atom, such as methylene, sub- second Base, propylidene or similar group.

Term " C5-C7 naphthenic base " refers to the naphthenic base with 5-7 carbon atom, such as cyclopenta, cyclohexyl or similar base Group.

Term " halogen " refers to F, Cl, Br and I.

Gene editing promotor

As used herein, " the compounds of this invention ", " formula A compound ", " gene editing promotor of the invention " are interchangeable Use, refer to structure shown in formula A compound or its pharmaceutically acceptable salt or its optical isomer or its racemic modification or Its solvate.It should be understood that the term further includes the mixture of said components.

In formula, each group is as defined above.

The compounds of this invention has extremely significant facilitation to the efficiency of gene editing

It in the present invention, further include the pharmaceutically acceptable salt of formula A compound.Term " pharmaceutically acceptable salt " refers to The compounds of this invention and acid or alkali are formed by the salt for being suitable as drug.Pharmaceutically acceptable salt includes inorganic salts and organic Salt.A kind of preferred salt is the salt that the compounds of this invention and acid are formed.The acid for suitably forming salt includes but is not limited to: hydrochloric acid, The inorganic acids such as hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, rich horse Acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzene methanesulfonic acid, the organic acids such as benzene sulfonic acid；With And the acidic amino acids such as aspartic acid, glutamic acid.

Formula A compound of the invention can be used method well known to those skilled in the art in the prior art and be prepared, right The response parameter of each step is not particularly limited.

As used herein, in formula A compound, if there is asymmetric carbon atom, then asymmetric carbon atom can be R configuration, May be S configuration, or both mixture.

Gene editing

The compounds of this invention is remarkably improved the efficiency of gene editing.

In the present invention, representative gene editing includes (but being not limited to): the gene editing based on CRISPR.It is typical Ground, the gene editing based on CRISPR include the gene editing based on CRISPR-Cpf1, the volume of the gene based on CRISPR-Cas Volume.Wherein, the Cas includes Cas9 etc..

A kind of preferred gene editing is CRISPR-Cpf1 gene editing.CRISPR-Cpf1 can be identified specifically and is rich in The PAM sequence (TTTN) of thymidylic acid (T) is expanded by the range of the RNA gene editing mediated.CRISPR-Cpf1 The cohesive end for generating 5nt, can start different DNA repair processes.The crRNA of the crRNA length ratio Cas9 of CRISPR-Cpf1 Much shorter, external synthesis are easier and are more suitable for polygenes editing process.The off-target rate of CRISPR-Cpf1 is relatively low.

Purposes

The combination of formula A compound of the invention and gene editing reagent (such as CRISPR-Cpf1 reagent), is remarkably improved base There are revolutionary potentiality because of the efficiency of editor, thus in different fields such as treatment uses.

Above-mentioned formula A compound of the invention can be used for improving the efficiency of the gene editing of CRISPR mediation, and then can be used for Prevention or treatment disease relevant to Disease-causing gene.

In one embodiment, the present invention provides a kind of gene editings for promoting CRISPR to mediate of external non-therapeutic Molecular sieve choosing method, comprising: the gene knock-in and medicament sifting motion system that CRISPR is mediated.

The present invention also provides a kind of gene editing method for promoting CRISPR to mediate using small molecule, this method be can be It is therapeutic or non-therapeutic.In general, the method comprising the steps of: applying formula A compound of the invention to the object of needs.

Preferably, the object includes people and non-human mammal (rodent, rabbit, monkey, domestic animal, dog, cat etc.).

Composition and method of administration

The present invention provides a kind of compositions of gene editing efficiency for promoting CRISPR to mediate.The composition Including (but being not limited to): pharmaceutical composition, scientific research reagent composition etc..

In the present invention, the composition can be directly used for promoting gene editing, for example, single-gene is knocked in, is dual-gene It knocks in, point mutation etc..

The present invention also provides a kind of pharmaceutical composition, it contains the compounds of this invention and pharmaceutically of safe and effective amount Acceptable carrier or excipient.This kind of carrier includes (but being not limited to): salt water, buffer, glucose, water, glycerol, second Alcohol, pulvis, and combinations thereof.Pharmaceutical preparation should match with administration mode.

By taking pharmaceutical composition as an example, composition of the invention can be made into injection form, such as with physiological saline or contain There are glucose and the aqueous solution of other adjuvants to be prepared by conventional method.The pharmaceutical composition of such as tablet and capsule etc Object can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule preferably aseptically manufacture. Pharmaceutical composition of the invention can also be made into pulvis for Neulized inhalation.

For pharmaceutical composition of the invention, required object (such as people and the inhuman food in one's mouth can be applied to by way of conventional Newborn animal).Representative method of application includes (but being not limited to): oral, injection, local application etc..

Main advantages of the present invention include:

(a) provide a kind of small molecule compound that can promote gene editing significantly for the first time, the compound for The hPSC gene knock-in editor that CRISPR-Cpf1/Cas9 is mediated is particularly effective.

(b) the present invention is based on the combinations of formula A compound and CRISPR-Cpf1, provide a kind of letter for accurate gene editing Single and efficient strategy.

(c) high-throughput screening method based on multipotential stem cell of the invention establishes a kind of drug sieve of no skewed popularity Select system.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

Universal method

Plasmid construction

For pCpfcr carrier, by BbsI enzyme recognition sequence and DR (forward direction repeats) sequence design in the upstream of U6 promoter In primer；U6 promoter region is passed through into PCR amplification using pUcm-T carrier cloning kit (Sangon Biotech company) Then it is cloned into carrier T.When constructing targeting sequence (crRNA) of the pCpfcr-crRNA carrier to express Cpf1, basis first The oligonucleotides of crRNA sequent synthesis 24bp, annealing form DNA double chain segment, and the pCpfcr for being cloned into BbsI digestion later is carried On body.

About building OCT4-tdTomato donor plasmid, using the method for PCR by OCT4-2A-mOrange donor plasmid (Addgene, Plasmid#66986) removes mOrange sequence as skeleton, and PCR amplification goes out tdTomato sequence later, uses TdTomato sequence is connected on skeleton by Gibson Assembly kit (New England Biolabs).All loads Body is sequenced by Sanger and is checked.

Cell culture

HPSCs is cultivated according to a conventional method.H1 embryonic stem cell line, MEL1 embryonic stem cell line and hiPSCs are cultivated with hPSC Base culture, consisting of: DMEM/F12 (Life Technologies), 20% serum deprivation sub KSR (Life Technologies), 1X nonessential amino acid (Life Technologies), 100X penicillin/streptomycin (Life Technologies), 0.055mM mercaptoethanol (Sigma) and 10ng/mL bFGF (Peprotech).

Every 3-6 days, hPSC cell is digested with Accutase (Life Technologies), and with the ratio of 1:3 to 1:6 Example passage.0.5 μM of Thiazovivin will be added in hPSC culture medium when each passage or defrosting cell and help stem cell Adherent survival.

Electricity turns

Prepare electricity turn after extracting plasmid with extraction reagent kit in ZymoPURE plasmid.Take first 81.82 μ L Solution I with 18,18μL Supplement Ⅰ(Human Stem CellKit 1, Lonza) it is mixed together with plasmid It is made into electricity and turns liquid.

Gene knock-in is tested, plasmid is by 3 μ g pcDNA3.1-hLbCpf1 (Addgene company, plasmid number # 69988), 3 μ g pCpfcr-crRNA and 4 μ g of OCT4-tdTomato donor or OCT4-2A-eGFP-PGK-Puro Donor (Addgene company, plasmid number #31938) composition.HPSC cell dissociation is taken 1 at individual cells with Accutase ×10⁶A cell electricity consumption turns liquid resuspension, and is turned with Amaxa electroporation (Lonza) electricity.After electricity turns, cell kind to six orifice plates A hole cultivate 2-3 days.Later, efficiency is knocked in streaming or the analysis of T7E I.

For establishing cloning experimentation, 500-2000 cell of inoculation to 10cm culture dish and is cultivated 4-7 days.Choose clone to use In amplification and storage.After 2 weeks, the cell line of expression tdTomato is identified with the methods of immunostaining, streaming, sequencing.

Transfection

10% fetal calf serum, the training of 100 × penicillin/streptomycin culture medium are added with DMEM (Life Technologies) Support human embryo kidney (HEK) 293T cell.When 293T cell density reaches 70%-80%, Lipofectamine is used^TM3000 transfection reagents (Invitrogen) mixing plasmid is transfected.Plasmid mixture is by 1 μ g pY016 (pcDNA3.1-hLbCpf1) and 1 μ g of PCpfcr-crRNA composition.After transfection 2-3 days, with I experimental analysis of T7E.

PFLP and T7E I is analyzed

2-3 days collection cells after transfection or electricity turn extract genome with the small extraction reagent kit of rapid gene group DNA (ZYMO). With the segment for having CRISPR action target spot in Taq polymerase (Vazyme) amplification gene group.

RFLP is tested, takes 2 μ L amplified productions to limit enzymic digestion, and run electrophoretic analysis with 2% Ago-Gel.It is right It is returned in the PCR product of point mutation, 100 μ L with DNA Clean&Concentrator-5 (ZYMO RESEARCH) kits It receives, uses 2% Ago-Gel to run electrophoretic analysis later with NcoI enzyme (New England Biolabs) digestion.Mutation rate is public Formula 100 × (b+c)/(a+b+c) is calculated, wherein a is represented not by the product band intensity of NcoI digestion, and b and c respectively represent enzyme Product band intensity after cutting.

T7E I is tested, 10 μ L amplified productions is taken to mix and match with NEB buffer 2 (New England Biolabs) At the system of 16 μ L of total volume.Mixed system is incubated for 5 minutes at 95 DEG C, then with 2 DEG C of speed per second is down to 85 DEG C, later with 0.1 DEG C of speed per second is slowly dropped to 25 DEG C, final 4 DEG C of incubations.I enzyme (New of T7E is added in 16 μ L reaction products England Biolabs) be made into the system of 20 μ L of total volume and be incubated for 30 minutes at 37 DEG C, later with 2.5% Ago-Gel Run electrophoretic analysis.Glue figure by JS-2000 Gel Imager (Peiqing Science&Techenology) shoot and by The analysis of ImageJ software.

Mutation rate formula 100 × (1-(1-(b+c)/(a+b+c))^1/2) calculate, wherein a is represented not by I digestion of T7E Product band intensity, b and c respectively represent the product band intensity after digestion.

Flow cytometry

One hPSC for having transfected Cpf1, OCT4-crRNA and OCT4-tdTomato carrier is seeded in six orifice plates Kong Zhong, with hPSC culture medium culture 3-4 days.Differentiation and undifferentiated cell Accutase are digested 3-5 minutes at single thin Born of the same parents.It is resuspended later with 1 × phosphate buffer of 0.3-1mL.Data finally are obtained with Beckman flow cytometer, and analyze knot Fruit.

HPSC differentiation

By about 200 undifferentiated OCT4-tdTomato hPSC cell inoculations in a hole of six orifice plates, it is used in combination HPSC culture medium culture 4-7 days.Change into later differential medium (DMEM/F12,20% serum substitute, 1 × NEAA, 100 × Penicillin/streptomycin (Life Techenologies), 0.055mM 2 mercapto ethanol (Sigma), 0.1 μM of LDN225,10 μM E616452 it) cultivates 3 days.After 4th or the 5th day, cell can use streaming and immunostained for analysis.

Immunostaining

It is cell 10-15 minutes fixed using 4% paraformaldehyde at room temperature, subsequent 1x PBST (1x PBS+0.3%Triton X-100 (Vetec)) it washes three times, five minutes every time.Next, cell is at confining liquid (1x PBST+5%BSA (fetal calf serum)) After middle incubation at room temperature 0.5-1 hours, primary antibody is incubated overnight (4 DEG C).Then, it is cleaned three times with PBST at room temperature, 15 minutes every time, Then corresponding secondary antibody is added, is incubated for 1 hour.Finally, being dyed using Hoechst (1:5000) to nucleus.

Apoptosis assay

Apoptosis is analyzed by apoptosis kit.DMSO, AZD-7762 (1 μM) and VE-822 (1 μ are used first M human pluripotent stem cells) are handled.Collect 5x10⁵A cell is simultaneously washed twice with ice PBS.Cell is centrifuged 5 minutes at 4 DEG C, then with 50 μ L 1x Binding Buffer is resuspended, and 2.5 μ L Annexin V (AV)-FITC and the incubation at room temperature of 2.5 μ L PI dyeing liquors are added 10-15 minutes.It is then added with the 1x Binding Buffer of 250 μ L into mixed liquor.Finally use flow cytomery Apoptosis.

It misses the target analysis

Cas-OFFinder can be used to detect site of potentially missing the target.The misfit value set is less than or equal to 6.It finds potential Site of missing the target after, design primer expands the site fragment that misses the target using PCR in cellular genome, later pass through Sanger be sequenced Method carries out sequencing identification.

Statistical analysis

Double tail t examine to obtain P value, and all statistics are indicated with mean ± s.e.

Embodiment 1.

Knockout cells system is efficiently constructed using CRISPR-Cpf1 in human pluripotent stem cells

In order to use CRISPR-Cpf1 gene editing system in human pluripotent stem cells, one is constructed by U6 promoter The crRNA expression plasmid (Fig. 4 a and 4b) of starting.

Pick several interested genes, including ALKBH1 and CLEC16A.ALKBH1 is tRNA demethylase, CLEC16A plays an important role in the generating process of diabetes.

In order to construct the crRNA plasmid of gene specific, the crRNAs (figure that a group-specific targets these genes is devised 1b and table 1).

[table 1]

The nucleic acid sequence of building expression crRNA plasmid

Construct the PCR primer of plasmid

PCR primer for genotype identification and sequencing

The PCR primer of T7EI experiment

The primer of RFLP experiment

The primer of qPCR

The PCR primer of off-target rate analysis

SsODN template sequence

In 293T cell, the genome edit capability of these crRNAs is tested.In T7E1 (T7 restriction endonuclease I) test, 20-30% insertion and deletion ratio (Fig. 4 c) is observed.

Then, the ability (Fig. 1 a) for the genome editor that CRISPER/Cpf1 is mediated in hPSCs is had studied.It is tried in T7E1 In testing, observe also there is 20-30% (figure to the ALKBH1 and CLEC16A efficiency for carrying out gene knockout in hESCs and hiPSCs 1c)。

HPSC system is knocked out in order to further establish, the hPSCs of transfection is passed on low cell density, picked clones, and (Fig. 1 a) is analyzed by the identification of pcr gene type.

For ALKBH1, monoallelic knockout is had occurred in 20.8% clone, and 12.5% has occurred diallele knockout (Fig. 1 e).

For CLEC16A, monoallelic knockout is had occurred in 25% clone, and double equipotential bases occur for 16.7% clone Because of knockout (Fig. 1 e).

Mulberry lattice sequencing result confirms, after gene knockout occurs in ALKBH1 and CLEC16A target site, genome sequence Number of base is lacked, to realize gene knockout (Fig. 1 f).

In addition, also being surveyed by PCR amplification and mulberry lattice for some sites of potentially missing the target found based on software analysis Sequence method is analyzed, as a result without the generation (Fig. 6,7b) of discovery miss target phenomenon.

To sum up, above-mentioned experimental result clearly demonstrates that CRISPR-Cpf1 can efficiently carry out base in hPSCs Because knocking out.

Embodiment 2.

Screening can remarkably promote the small molecule of the HDR of CRISPR-Cpf1 mediation in hPSCs

In the present embodiment, for the ability for examining CRISPR-Cpf1 to carry out gene insertion, using the method that electricity turns by three A plasmid is transferred in hPSCs: plasmid expression Cpf1, the specific crRNA containing targeting OCT4, and includes eGFP The HDR template plasmid (Fig. 8 a) of reporter gene and puromycin (Puro) resistance.After electricity turns, first with ordinary culture medium culture The two days time of hPSCs, backward culture medium in be added puromycin handle 3-4 days time.It observes and is carried out by HDR mode The relatively inefficient and needs of genome reparation are further improved.

In addition, the SCR7 compound conduct pair of the gene editing efficiency of CRISPR-Cas9 mediation can be improved known to According to compound.

As a result:

Experiment shows SCR7 (small molecule of the known gene knock-in that can promote CRISPR-Cas9 mediation), in CRISPR- Without significant facilitation effect (Fig. 2 e) in the gene knock-in that Cpf1 is mediated.

This shows that CRISPR-Cpf1 system and CRISPR-Cas9 system have some differences in practical applications.Therefore, It was found that small molecule compound that is new and being effectively facilitated HDR is highly important.

Embodiment 3

Chemical small molecule screening

In the present embodiment, for hundreds of candidate compound, is screened and tested one by one.

In the present embodiment, in order to improve the efficiency of the gene editing that in hPSCs CRISPR-Cpf1 is mediated, with tool There is the OCT4-eGFP of puromycin to knock in screening system and has carried out extensive screening compound (Fig. 2 a).

Specifically, three plasmids are transferred in hPSCs using the method that electricity turns: a plasmid expression Cpf1, one contains Target the specific crRNA of OCT4, and the HDR template plasmid comprising eGFP reporter gene and puromycin (Puro) resistance (Fig. 8 a).

100 μ L cells need to be added in each hole of 48 orifice plates before electricity turns operation for each candidate compound Culture medium and the small molecule (1:1000) of 0.2 μ L screening.After cell electroporation, 1x10⁶A cell mixing is being added to In the 4.8mL hPSC culture medium of Thiazovivin small molecule, it is seeded in 48 orifice plates.Using being added to small point of VE-822 The culture medium culture cell of son stops adding VE-822 small molecule two days later, and the puromycin of 1 μ g/mL is added in the medium Continue culture 3-5 days.Later, the clone with puromycin-resistant screened is counted, to detect small molecule Efficiency.

As a result:

About 600 small molecules, small molecular VE-822 in kinases library have been screened in total improves hPSC grams very significantly Grand quantity (Fig. 2 b, 2c, 2e, 2f, 3b, 3c, 8d, 10b, 10e and 11a), this prompt compound VE-822 are remarkably improved gene Editorial efficiency.

Embodiment 4

The compliance test result of gene editing promotor

In the present embodiment, the function and effect of further progress experimental verification VE-822.Test method is the same as embodiment 3.

The result shows that VE-822 reaches its maximum effect effect (Fig. 2 d) under 1 μM.VE-822 is the suppression of ATR kinases Preparation can promote gene knock-in efficiency to improve 5.9 times.

In addition, VE-822 does not significantly improve the cell proliferation rate of hPSCs, show that VE-822 is not by promoting cell Proliferation improves gene knock-in efficiency (Fig. 9 a).

In addition, VE-822 does not show the cytotoxicity (Fig. 9 c and 9d) to hPSCs.The hPSCs handled with VE-822 Still high these special versatility genes (Fig. 9 b) of expression OCT4 and NANOG.

In contrast, in the case where knocking in the hPSCs that CRISPR-Cpf1 is mediated, control compound SCR7 is not shown The facilitation of work, but the gene knock-in (Fig. 2 e and 8d) that VE-822 can promote Cpf1 to mediate significantly in hPSCs.

In CRISPR-Cas9 system, the efficiency that SCR7 is improved is lower than 2 times, and the function and effect of VE-822 are more significant (about 4 times).

In addition, striking the gene knock-in efficiency that low ATR gene can promote CRISPR-Cpf1 to mediate with siRNAs, illustrate VE- 822 work (Fig. 2 g) by targeting ATR really.The above results successfully confirm that VE-822 can be improved significantly in hPSCs The gene knock-in efficiency that middle CRISPR-Cpf1 is mediated.

Embodiment 5.

The generation for the hPSCs gene knock-in cell line that small molecule promotes CRISPR-Cpf1 to mediate significantly

In the present embodiment, CRISPR-Cpf1 is further had detected for generating hPSCs in no drug screening The ability of gene knock-in cell line.

Method is as follows: 3 plasmid electricity are rotated into hPSCs: a plasmid expression Cpf1, one comprising selectively targeted The crRNA of OCT4, and a donor plasmid (Fig. 3 a) containing OCT4-2A-tdTomato.After 5-6 days, tdTomato is observed Positive cell passes through the efficiency of FACS (streaming) analysis detection gene knock-in.

As the result is shown: VE-822 can improve the percentage of tdTomato positive cell significantly, and this demonstrate VE-822 It can promote gene knock-in (Fig. 3 b, c) of the CRISPR-Cpf1 in hPSCs.

Later, the several tdTomato positive colonies of picking carry out the foundation of hPSC cell line.The result of PCR analysis is aobvious Show and successfully incorporates tdTomato reporter gene (Fig. 3 f) in these cell lines.These hPSC cell lines built up coexpression TdTomato and versatility gene OCT4 (Fig. 3 d).

After the differentiation of these stem cells, cell loss multipotency gene can be proved by immunostaining and FACS experiment The expression of OCT4, meanwhile, cell also no longer expresses tdTomato fluorescent reporter gene (Fig. 3 d, e).Therefore, OCT4-tdTomato It can be in the expression of hPSCs stage reflection endogenous versatility gene OCT4 synchronous with during differentiation.

Later, it is sequenced using sanger method.Sequencing result prompt, in these OCT4-tdTomato cell lines, 2A- TdTomato sequence is successfully plugged into genome, and the termination codon subsequence of OCT4 gene is substituted.

In addition, carrying out dual-gene knock in the experiment proves that VE-822 can be shown to two gene locis of OCT4 and ALBUMIN Write the dual-gene efficiency (Figure 11 a) knocked in for promoting CRISPR-Cpf1 to mediate.

For mediating the point mutation generated to test (Fig. 3 h) using CRISPR-Cpf1, method is into hPSC cell Electricity is transferred to carrier, ALKBH1-crRNA carrier and the single strand nucleotide sequence template of expression Cpf1.Candidate small molecule group as the result is shown The efficiency (Fig. 3 i and 3j) that point mutation is constructed using single-chain nucleic acid as template can be remarkably promoted by closing.

Above-mentioned test result shows that the combination based on CRISPR-Cpf1 and small molecule can carry out effectively in hPSCs Gene insertion.

It discusses

In the present invention, the present invention is proved by test, the gene editing based on CRISPR-Cpf1, can be dry in people's multipotency Efficient gene knockout and inserting edition are carried out in cell line.T7EI the results show CRISPR-Cpf1 success is in ALKBH1 Genome cutting has been carried out on the specific site of two genes of CLEC16A.Gene is carried out by the method for choosing monoclonal later Type identification, this gene knockout efficiency is higher as the result is shown.

By the result of sequencing also it is concluded that CRISPR-Cpf1 can smoothly construct the cell line of gene knockout.

For gene loading test, the present inventor constructs the carrier of OCT4-tdTomato, is situated between by CRISPR-Cpf1 The gene editing technology led successfully is that OCT4 gene inserts red fluorescence reporter gene, and by flow cytometry and exempts from Epidemic disease fluorescent staining experiment can detecte the hPSCs for being successively inserted into fluorescent reporter gene.

In addition, the crRNA length for being unique in that it of CRISPR-Cpf1 is shorter, off-target rate is lower, these characteristics make It has very high application value.But use current gene editing tool progress lower with the efficiency of source orientation reparation, consumption When it is longer.

In view of the specificity of CRISPR-Cpf1 and CRISPR-Cas9, a feasible Chemical Screening system, identification are constructed It is very significant to some small molecule compounds for improving gene editing efficiency.

It is noted that the present inventor is that the high flux screening of human pluripotent stem cells establishes a kind of medicine of no skewed popularity Object screening system, and this unique screening system can make the inventors discovered that some novel and interesting small molecule chemical combination Object, thus the accurate gene editing process for promoting CRISPR-Cpf1 to mediate.

The present inventor carries out gene insertion using OCT4-eGFP-Puro carrier in hPSCs, utilizes puromycin later (Puro) drug screening is carried out to cell, final to can get the monoclonal with Puro resistance, the present inventor makees monoclonal number It is output result the difference that compares control group and each experimental group.The present inventor has been screened in total close to 600 chemical small molecules, The hPSC gene editing process that small molecule compound VE-822 can promote CRISPR-Cpf1 to mediate significantly is finally found that.

Gene knock-in efficiency can be improved 5.9 times after small molecule VE-822 is added, and the function of the small molecule is significantly excellent In some chemical small molecules previously having had found.Importantly, the group of CRISPR-Cpf1 and small molecule compound is combined into accurately Gene editing provides one kind simply and efficient strategy, can also further develop and interior raw with the mankind applied to animal body Gene editing in cell colonization.Unquestionably, these progress will provide new Method and kit for for genome project, accelerate the mankind The development of disease treatment new method.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Zhejiang University

<120>promote the screening compound of gene editing and identify and its apply

<130> P2017-2499

<160> 89

<170> PatentIn version 3.5

<210> 1

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

tttccccctg tctccgtcac cactctgg 28

<210> 2

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

agatcccctg tctctgtcac cactctgg 28

<210> 3

<211> 28

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<213>artificial sequence (Artificial Sequence)

<400> 3

aaaaccagag tggtgacaga gacagggg 28

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<400> 4

ccctgtctcc gtcaccactc tgg 23

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<211> 25

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caccgccctg tctccgtcac cactc 25

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<400> 6

aaacgagtgg tgacggagac agggc 25

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<400> 7

tgagcccggg gaggacgcct ttcggaaa 28

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agatcgaaag gcgtcctccc cgggctca 28

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<400> 9

aaaatgagcc cggggaggac gcctttcg 28

<210> 10

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<400> 10

tttgccccgc gtcttggagg gacttacg 28

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<400> 11

agatccccgc gtcttggagg gacttacg 28

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<400> 12

aaaacgtaag tccctccaag acgcgggg 28

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cccactaacc gtcacactta gcaataaa 28

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agatttgcta agtgtgacgg ttagtggg 28

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aaaacccact aaccgtcaca cttagcaa 28

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tttcacctcc agaccgattg ttattgaa 28

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agatacctcc agaccgattg ttattgaa 28

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aaaattcaat aacaatcggt ctggaggt 28

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tttcggaaac ttttccgctt ctaccgtc 28

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<400> 20

agatggaaac ttttccgctt ctaccgtc 28

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aaaagacggt agaagcggaa aagtttcc 28

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tttgaccaaa aacaccacag tcacagaa 28

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agataccaaa aacaccacag tcacagaa 28

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aaaattctgt gactgtggtg tttttggt 28

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gccttaggct tataacatca catttaaa 28

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agataatgtg atgttataag cctaaggc 28

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aaaagcctta ggcttataac atcacatt 28

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<400> 28

ccttaggctt ataacatcac att 23

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caccgaatgt gatgttataa gccta 25

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<400> 30

aaactaggct tataacatca cattc 25

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<400> 31

gagggcctat ttcccatgat tcct 24

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<213>artificial sequence (Artificial Sequence)

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aaaaaaaggt cttctcgaag acccatctac acttagtaga aattcggtgt ttcgtccttt 60

ccacaag 67

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<212> DNA

<213>artificial sequence (Artificial Sequence)

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ggtgcctgcc cttctaggaa 20

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aggaccgggg ttttcttcca 20

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tggaagaaaa ccccggtcct atggtgagca agggcgagga 40

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ttcctagaag ggcaggcacc ttacttgtac agctcgtcca 40

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atcttcagga ggtaagggtg 20

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cgatctcgaa ctcgtggc 18

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gcttccatca ctggctcgta 20

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cgacatcccc tgcttgtttc 20

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<213>artificial sequence (Artificial Sequence)

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aaattccact actcccacat ctcc 24

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<213>artificial sequence (Artificial Sequence)

<400> 42

cggattccca ggctcttttg 20

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acccttcaaa gcattgtctg c 21

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aacttcccca tttttggctt g 21

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atgtccgtga gcttccgtc 19

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cgatctcgaa ctcgtggc 18

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gatcaagcag cgactatgca 20

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tcacttgggt atgagcattg 20

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aaattccact actcccacat ctcc 24

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<211> 20

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cggattccca ggctcttttg 20

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acccttcaaa gcattgtctg c 21

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aacttcccca tttttggctt g 21

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aaattccact actcccacat ctcc 24

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cggattccca ggctcttttg 20

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acccttcaaa gcattgtctg c 21

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aacttcccca tttttggctt g 21

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atatgaagcg tgccgtagac t 21

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tgcctatgtc tggctctatt ctg 23

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ggccaaaggc agttgtattg a 21

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gtgagtaccc caaaaatagc agg 23

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<400> 61

tgcaccacca actgcttagc 20

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ggcatggact gtggtcatga g 21

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<400> 63

ggcgtgtcac aagaaggg 18

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<400> 64

ggaggcggag gatgaagt 18

<210> 65

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tgtgggtgac agaatggt 18

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aatgggtcct gcaagtaa 18

<210> 67

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cacgccaaag ccttcata 18

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gaacggatgg gacggtag 18

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ttaggcagac ctctgtga 18

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tgggcaacaa gagcaaag 18

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<400> 71

attgaaacgc cacagatt 18

<210> 72

<211> 18

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<400> 72

ctagccctta gccttgac 18

<210> 73

<211> 19

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<400> 73

cgtatttcag cgtttgttc 19

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<400> 74

gctaagcgag atttcctaa 19

<210> 75

<211> 18

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<400> 75

ccctaaatgg agcgagaa 18

<210> 76

<211> 18

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<400> 76

gtagccttgg gaaacagc 18

<210> 77

<211> 18

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<400> 77

cataaggtcg ggtcaggg 18

<210> 78

<211> 18

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<213>artificial sequence (Artificial Sequence)

<400> 78

aagaaagcac caaggcac 18

<210> 79

<211> 20

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<400> 79

acagagtatc gcaggcacaa 20

<210> 80

<211> 20

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<400> 80

cctttccacc cacccttgat 20

<210> 81

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 81

gtgactgttt ccctttccct ct 22

<210> 82

<211> 20

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<213>artificial sequence (Artificial Sequence)

<400> 82

tgtcatgggg aattgctggt 20

<210> 83

<211> 18

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<213>artificial sequence (Artificial Sequence)

<400> 83

gaatttaggt gaagggta 18

<210> 84

<211> 18

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<400> 84

acaagtcgag ctgataca 18

<210> 85

<211> 21

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<400> 85

gttgtcagga aatgcagagc c 21

<210> 86

<211> 20

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<213>artificial sequence (Artificial Sequence)

<400> 86

agtcctacgt ccgagagtga 20

<210> 87

<211> 21

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<400> 87

acatattcaa caggtgccag c 21

<210> 88

<211> 21

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<400> 88

aagtctcatg ggttttgggg g 21

<210> 89

<211> 120

<212> DNA

<213>homo sapiens (Homo sapiens)

<400> 89

gcgagatggg gaagatggca gcggccgtgg gctctgtggc gactctggcg actgagccca 60

tggaggacgc ctttcggaaa cttttccgct tctaccgtca gagccggccc gggaccgcag 120

Claims

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；

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-；

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；

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, 0,1 or 2 p；

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

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, 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, each L2 independently is :-SO₂-；

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

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

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, 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

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

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 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；

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)；

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.