CN110241135B

CN110241135B - Screening and identification of compounds that promote gene editing and uses thereof

Info

Publication number: CN110241135B
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: 2022-12-23
Anticipated expiration: 2038-03-09
Also published as: CN110241135A

Abstract

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

Description

Screening and identification of compounds that promote gene editing and uses thereof

Technical Field

The present invention relates to the field of biology, more specifically to screening and identification of compounds that facilitate gene editing and uses thereof.

Background

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

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

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

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

The gene editing technology based on CRISPR-Cpf1 nuclease is another oriented gene editing technology, the range of gene editing is enlarged, and the precision is higher, however, the gene editing efficiency of CRISPR-Cpf1 is difficult to satisfy. In addition, chemical small molecules that can facilitate CRISPR-Cpf1 gene editing have not been reported.

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

Disclosure of Invention

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

In a first aspect of the invention, there is provided a use of a compound represented by formula a, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof, for preparing an accelerator or a formulation for accelerating gene editing;

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

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

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

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 cycloalkyl;

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 cycloalkyl;

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 cycloalkyl, - (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 cycloalkyl;

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

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 combinations 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 said cycloheteroalkyl is 5-7 membered and contains 1-3 heteroatoms selected from the group consisting of N, O, and S;

in another preferred embodiment, each R1 is independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb);

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

in another preferred embodiment, each R2 is independently selected from the group consisting of: 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 embodiment, R3 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb);

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

In another preferred embodiment, 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 group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, -N (Ra) (Rb);

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

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

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

in another preferred embodiment, each L2 is independently selected from the group consisting of: -SO ₂ -, -NH-, -O-, or combinations thereof;

in another preferred embodiment, ra and Rb are each independently selected from the group consisting of: H. substituted or unsubstituted C1-C3 alkyl;

in another preferred embodiment, each R1 is independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C3 alkyl;

in another preferred embodiment, n is 0 or 1;

in another preferred embodiment, each R2 is independently selected from the group consisting of: 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 embodiment, R3 is selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C3 alkyl, -N (Ra) (Rb);

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

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

in another preferred embodiment, R6 is selected from the group consisting of: 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 group consisting of: H. halogen, substituted or unsubstituted C1-C3 alkyl, -N (Ra) (Rb);

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

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

In another preferred embodiment, ra and Rb are each independently selected from the group consisting of: H. unsubstituted C1-C3 alkyl;

in another preferred embodiment, 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 embodiment, the compound is

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

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

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

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

in another preferred embodiment, the sample is from animal, plant, microorganism (including bacteria, virus);

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

in another preferred embodiment, the kit further comprises instructions;

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

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

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

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

in another preferred embodiment, the gene editing is directed to pathogenic genes, tumor-associated genes (e.g., oncogenes), immune-related genes (e.g., genes associated with autoimmunity), 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 invention, there is provided a kit comprising:

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

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

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

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

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

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

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For the sake of space, it is not described here in a single word.

Drawings

FIG. 1 shows that CRISPR-Cpf1 efficiently generated knockout hPSC cell lines. FIG. 1a shows the experimental scheme flow for obtaining knockout human pluripotent stem cell lines. FIG. 1b shows a schematic representation of Cpf1 crRNA at the ALKBH1 and CLEC16A targeting sites, including exons, PCR amplicons and cleavage sites for PCR analysis, the crRNA targeting sequence being shown in bold and the PAM sequence being grey. Fig. 1c shows the efficiency of T7EI experiments to detect crRNA targeting ALKBH1 and CLEC16A in MEL1 human pluripotent stem cells, indel frequency calculated using the expected fragment. FIG. 1d shows T7EI detection of ALKBH1 crRNA in H1 type hESCs and hipSCs. Indel frequencies are calculated using the correlation bands. FIG. 1e shows PCR analysis after transfection of crRNA. For ALKBH1, two crRNAs are transfected together. Figure 1f shows the sequencing results of gene editing sites in the ALKBH1 and CLEC16A knocked-out human pluripotent stem cell line.

Figure 2 shows the identification of small molecules capable of significantly facilitating CRISPR-Cpf 1-mediated knock-in of genes in human pluripotent stem cells. Figure 2a shows a chemical screening protocol. Figure 2b shows the results of the chemical screening. Screening about 600 small molecules, and calculating the number of puromycin resistant cell clusters. The small molecules above the line are further identified candidate chemical small molecules. The mean cell mass number of DMSO was 2. Figure 2c shows the results of repeated experiments with candidate small molecules. VE-822 and AZD-7762 are the two small molecules that increase the number of puromycin resistant cell aggregates the most. FIG. 2d shows the optimal working concentration test for VE-822 and AZD-7762. FIG. 2e shows the promoting effect of VE-822, AZD-7762 and SCR7 on CRISPR-Cpf1 mediated gene knock-in human pluripotent stem cells. FIG. 2f shows the effect of testing VE-822 and AZD-7762 in H1 human embryonic stem cell lines and induced human pluripotent stem cell lines. Fig. 2g shows the use of siRNA to knock down ATR and CHEK1 in human pluripotent stem cells, observing its effect on CRISPR-Cpf1 mediated gene knock-out.

Fig. 3 shows the establishment of small molecules that significantly facilitate CRISPR-Cpf 1-mediated knock-in of hPSC gene into cell lines. FIG. 3a shows the targeting site information of OCT 4-2A-tdTomato. FIG. 3b shows FACS analysis of OCT4-2A-tdTomato positive cells after transfection of OCT4-2A-tdTomato plasmid and OCT4 targeting crRNA plasmid into human pluripotent stem cells. FIG. 3c shows that VE-822 and AZD-7762 increased the percentage of OCT4-2A-tdTomato positive cells. FIG. 3d shows the identification of OCT4-tdTomato reporter cell line. FIG. 3e shows FACS analysis of OCT4-tdTomato reporter cell line. FIG. 3f shows the PCR identification of OCT4-tdTomato reporter cell line. The expected band size is 1131bp. FIG. 3g shows the sequencing results of the positions of the wild-type and inserted reporter gene in OCT4-tdTomato reporter cell line. Fig. 3h shows the ssODN-mediated gene editing scheme. Point mutations were introduced using ALKBH1-cr1 and synthetic 120-nt ssODN template and detected by RFLP method. FIG. 3i shows the RFLP detection results. The NcoI cleavage site was used to identify the efficiency of the ssoDN-mediated knock-in. FIG. 3j shows that the efficiency of SSODN-mediated genome editing by VE-822 and AZD-7762 was increased 3-fold over the control group. Figure 3k shows the results of the algbh 1 site Sanger sequencing before and after modification.

Figure 4 shows a working model of CRISPR-Cpf 1. Fig. 4a shows a schematic diagram of the gene editing system of CRISPR-Cpf 1. FIG. 4b shows the construction of pCpfcr plasmid. The direct repetitive sequence, the BbsI recognition sequence and the poly-T sequence are sequentially arranged behind the U6 promoter. Fig. 4c shows the efficiency of editing for detecting crRNA targeting ALKBH1 and CLEC16A in 293T cells using T7 EI.

FIG. 5 shows an identification model for CRISPR-Cpf1 generation knock-out hPSC cell lines. FIG. 5a shows crRNA cr1 and cr2 knock-out ALKBH1 using two ALKBH1 s. If the two crRNAs are cleaved successfully, a deletion of about 190bp occurs at the ALKBH1 site. FIG. 5b shows a schematic diagram of the results of the PCR genotyping. The band in the homozygous for ALKBH1 knock-out is 471bp, and the band in WT clone is 664bp. In heterozygotes, we observed two bands around 471bp and 664bp, as well as an additional hybridizing band. FIG. 5c shows a schematic representation of a hybrid band. FIG. 5d shows the Sanger sequencing results of the hybridization bands. FIG. 5e shows the results of experimental identification of T7EI in the hybridization bands. T7EI can specifically cut hybrid bands. FIG. 5f shows a pattern of identifying CLEC16A site knockouts using RFLP. The CLEC16A-cr1 targeting site comprises a Tsp45I cleavage site. FIG. 5g shows a schematic representation of RFLP results. The Tsp45I cleavage site in the WT cell line was intact, and two bands (220 bp and 296 bp) appeared after cleavage. Indes appeared at the Tsp45I site in the homozygote of the CLEC16A knockout, resulting in a band of 516bp. Three bands (220 bp, 296bp and 516 bp) appear in heterozygotes.

Fig. 6, 7 show potential off-target site analysis for CRISPR-Cpf 1. FIG. 6a shows details of predicting potential off-target sites for ALKBH1-cr 2. Figure 6b shows Sanger sequencing results of potential off-target sites. Potential off-target sites were amplified by PCR and sequenced by Sanger sequencing. FIG. 7a shows detailed information predicting potential off-target sites for CLEC16A-cr 1. Figure 7b shows Sanger sequencing results of potential off-target sites. Potential off-target sites were amplified by PCR and sequenced by Sanger sequencing.

Fig. 8 shows that the candidate small molecule can significantly promote CRISPR-Cpf1 or CRISPR-Cas9 mediated hPSC gene knock-in at the OCT4 site. Fig. 8a shows a schematic diagram of targeting the OCT4 site with CRISPR-Cpf 1. The OCT4crRNA targets a sequence downstream of the PAM sequence. Figure 8b shows the results of Sanger sequencing at the OCT4 site target. Figure 8c shows a schematic diagram of targeting the OCT4 site using CRISPR-Cas 9. Figure 8d shows a test of the effect of VE-822, AZD-7762, and SCR7 on CRISPR-Cas9 mediated gene knock-in human pluripotent stem cells.

Figure 9 shows toxicity testing of candidate small molecules. FIG. 9a shows a statistical plot of the number of human pluripotent stem cells after treatment with DMSO, AZD-7762 and VE-822. FIG. 9b shows an immunostaining pattern of VE-822 and AZD-7762 treated human pluripotent stem cells. Figure 9c shows FACS results for apoptosis. FIG. 9d shows the determination of the proportion of apoptosis in AZD-7762 and VE-822 treated cells.

FIG. 10 shows that candidate small molecules can significantly facilitate CRISPR-Cpf 1-or CRISPR-Cas 9-mediated hPSC gene knock-in at ALBUMIN sites. Figure 10a shows a schematic diagram of targeting an alomin site with CRISPR-Cpf 1. FIG. 10b shows the effect of VE-822 and AZD-7762 in CRISPR-Cpf 1-mediated knock-in of the ALBUMIN site. FIG. 10c shows the results of Sanger sequencing at the target site of the ALBUMIN site. Figure 10d shows a schematic diagram of targeting the ALUMIN site using CRISPR-Cas 9. Figure 10e shows the role of VE-822 and AZD-7762 in CRISPR-Cas9 mediated knock-in of the ALBUMIN site. FIG. 10f shows the results of Sanger sequencing at the target site of the ALBUMIN site.

Fig. 11 shows that the candidate small molecules can significantly promote CRISPR-Cpf 1-mediated knockin of hPSC double genes. FIG. 11a shows the effect of VE-822 and AZD-7762 in CRISPR-Cpf1 mediated double knock-in experiments at the OCT4 and ALBUMIN sites. FIG. 11b shows photographs of representative tdTomato-positive anti-Puro cell masses in a double knock-in experiment. FIG. 11c shows Sanger sequencing results at the target site of OCT4 and ALBUMIN sites.

Detailed Description

The inventor of the invention, after extensive and intensive research, unexpectedly discovers that a compound with a structure shown as formula A can significantly improve the gene editing efficiency of CRISPR, and especially can significantly promote the gene knock-in efficiency mediated by CRISPR-Cpf 1. Experiments show that the compound of the formula A (such as VE-822) can remarkably promote CRISPR-Cpf 1-mediated gene editing efficiency, for example, high-efficiency gene editing is carried out on human pluripotent stem cells so as to generate a gene knock-in cell line. On this basis, the inventors have completed the present invention.

Term(s) for

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

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

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

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

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

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

Gene editing promoter

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

In the formula, each group is as defined above.

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

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

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

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

Gene editing

The compound can obviously improve the efficiency of gene editing.

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

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

Use of

The combination of the compound of formula A and the gene editing reagent (such as CRISPR-Cpf1 reagent) can obviously improve the efficiency of gene editing, thereby having revolutionary potential in different fields such as therapeutic application and the like.

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

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

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

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

Compositions and methods of administration

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

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

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

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

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

The main advantages of the present invention include:

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

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

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

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are generally performed 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's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

General procedure

Plasmid construction

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

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

Cell culture

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

Every 3-6 days, hPSC cells were digested with Accutase (Life Technologies) and passaged at a rate of 1:3 to 1:6. Each passage or thawing of the cells 0.5 μ M Thiazovivin was added to the hPSC medium to help stem cell adherent survival.

Electric rotating device

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

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

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

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

Transfection

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

PFLP and T7EI assays

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

For RFLP experiments, 2. Mu.L of the amplification product was digested with restriction enzymes and analyzed by 2% agarose gel electrophoresis. For point mutations, 100. Mu.L of PCR product was recovered by purification using 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), wherein a represents the band intensity of the product not digested with NcoI, and b and c represent the band intensity of the product after digestion, respectively.

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

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

Flow cytometry

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

hPSC differentiation

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

Immunostaining

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

Apoptosis assay

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

Off-target analysis

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

Statistical analysis

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

Example 1.

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

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

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

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

[ TABLE 1 ]

Construction of nucleic acid sequences for expression of crRNA plasmids

PCR primers for plasmid construction

PCR primers for genotyping and sequencing

PCR primer for T7EI experiment

Primer for RFLP experiment

Primers for qPCR

PCR primer for off-target rate analysis

ssODN template sequences

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

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

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

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

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

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

In addition, for some potential off-target sites found based on software analysis, PCR amplification and Sanger sequencing were also used for analysis, and as a result, no off-target phenomenon was found (FIGS. 6 and 7 b).

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

Example 2.

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

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

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

As a result:

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

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

Example 3

Chemical small molecule screening

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

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

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

For each candidate compound, 100 μ L of cell culture medium and 0.2 μ L of the selected small molecule (1. After electroporation of the cells, 1X10 ⁶ The cells were mixed well in 4.8mL of hPSC medium supplemented with Thiazovivin small molecules and plated in a 48-well plate. Culturing the cells by using the culture medium added with the VE-822 micromolecules, stopping adding the VE-822 micromolecules after two days, adding 1 mu g/mL puromycin in the culture medium, and continuing culturing for 3-5 days. Subsequently, the efficiency of the small molecules was examined by counting the puromycin resistant clones selected.

As a result:

in total, about 600 small molecules of the kinase pool were screened, with the small molecule VE-822 very significantly increasing the number of hPSC clones (FIGS. 2b,2c,2e,2f,3b,3c,8d,10b,10e and 11 a), suggesting that the compound VE-822 may significantly increase gene editing efficiency.

Example 4

Effect verification of Gene editing promoters

In this example, the effect of VE-822 was further verified by experiments. The test method was the same as in example 3.

The results show that VE-822 reaches its maximum effect at 1. Mu.M (FIG. 2 d). VE-822 is an inhibitor of ATR kinase, and can improve gene knock-in efficiency by 5.9 times.

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

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

In contrast, the control compound SCR7 did not significantly promote CRISPR-Cpf 1-mediated knock-in hPSCs, but VE-822 could significantly promote Cpf 1-mediated gene knock-in hPSCs (fig. 2e and 8 d).

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

Furthermore, knockdown of the ATR gene with siRNAs could promote CRISPR-Cpf 1-mediated gene knock-in efficiency, suggesting VE-822 does act by targeting ATR (fig. 2 g). The results successfully confirm that VE-822 can significantly improve CRISPR-Cpf1 mediated gene knock-in efficiency in hPSCs.

Example 5.

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

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

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

The results show that: VE-822 was able to significantly increase the percentage of tdTomato positive cells, indicating that VE-822 could promote CRISPR-Cpf1 knock-in hPSCs (FIG. 3b, c).

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

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

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

In addition, double gene knock-in experiments performed on the two gene loci of OCT4 and ALBUMIN demonstrated that VE-822 can significantly promote the efficiency of CRISPR-Cpf 1-mediated double gene knock-in (FIG. 11 a).

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

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

Discussion of the related Art

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

Sequencing results can also conclude that the CRISPR-Cpf1 can successfully construct a gene knockout cell line.

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

In addition, the CRISPR-Cpf1 is unique in that the crRNA is shorter in length and lower in off-target rate, and the characteristics enable the CRISPR-Cpf1 to have high application value. However, homology-directed repair using current gene editing tools 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 a plurality of small molecular compounds for improving the gene editing efficiency.

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

The present inventors have used OCT4-eGFP-Puro vector to perform gene insertion into hPSCs, followed by drug screening of cells with puromycin (Puro), and finally obtained Puro-resistant monoclones. The inventor screens nearly 600 chemical small molecules in total, and finally discovers that the small molecule compound VE-822 can remarkably promote the CRISPR-Cpf 1-mediated hPSC gene editing process.

The gene knock-in efficiency can be improved by 5.9 times after the small molecule VE-822 is added, and the function of the small molecule is remarkably superior to that of some chemical small molecules which are found previously. Importantly, the combination of CRISPR-Cpf1 and small molecule compounds provides a simple and efficient strategy for precise gene editing, and can be further developed and applied to gene editing in animals and human germ cells. These advances will undoubtedly provide new methods and tools for genome engineering, accelerating the development of new methods for the treatment of human diseases.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents will fall within the scope of the invention as defined in the appended claims.

Sequence listing

<110> Zhejiang university

<120> screening and identification of compounds promoting gene editing and use thereof

<130> P2017-2499

<160> 89

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<213> Artificial Sequence (Artificial Sequence)

<400> 65

tgtgggtgac agaatggt 18

<210> 66

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

aatgggtcct gcaagtaa 18

<210> 67

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

cacgccaaag ccttcata 18

<210> 68

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

gaacggatgg gacggtag 18

<210> 69

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

ttaggcagac ctctgtga 18

<210> 70

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

tgggcaacaa gagcaaag 18

<210> 71

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

attgaaacgc cacagatt 18

<210> 72

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

ctagccctta gccttgac 18

<210> 73

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

cgtatttcag cgtttgttc 19

<210> 74

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

gctaagcgag atttcctaa 19

<210> 75

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

ccctaaatgg agcgagaa 18

<210> 76

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

gtagccttgg gaaacagc 18

<210> 77

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

cataaggtcg ggtcaggg 18

<210> 78

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

aagaaagcac caaggcac 18

<210> 79

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

acagagtatc gcaggcacaa 20

<210> 80

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<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

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

tgtcatgggg aattgctggt 20

<210> 83

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

gaatttaggt gaagggta 18

<210> 84

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

acaagtcgag ctgataca 18

<210> 85

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

gttgtcagga aatgcagagc c 21

<210> 86

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

agtcctacgt ccgagagtga 20

<210> 87

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

acatattcaa caggtgccag c 21

<210> 88

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

aagtctcatg ggttttgggg g 21

<210> 89

<211> 120

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 89

gcgagatggg gaagatggca gcggccgtgg gctctgtggc gactctggcg actgagccca 60

tggaggacgc ctttcggaaa cttttccgct tctaccgtca gagccggccc gggaccgcag 120

Claims

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

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

3. A method of promoting CRISPR-based gene editing in a cell in vitro, comprising:

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

wherein the gene editing promoter is the compound VE-822 of claim 1, or a pharmaceutically acceptable salt thereof, and the method is non-diagnostic and non-therapeutic.

4. The method of claim 3, wherein in step (a) the gene editing promoter is contacted with the cell undergoing gene editing before, during and/or after gene editing has occurred.

5. The method of claim 3, wherein the concentration of the gene-editing facilitator in the in vitro reaction system is 0 μ M to 2 μ M.

6. The method of claim 3, wherein in step (a), comprising:

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

7. A reagent product or combination of reagents comprising:

(i) A first agent which is a gene editing promoter which is compound VE-822 of claim 1, or a pharmaceutically acceptable salt thereof; and

(ii) A second agent that performs CRISPR gene editing.

8. Use of the reagent product of claim 7 for the preparation of a kit for performing CRISPR-based gene editing.

9. A kit, comprising:

(i) A first container, and a first agent in said first container, said first agent being a gene editing promoter, said gene editing promoter being compound VE-822 of claim 1, or a pharmaceutically acceptable salt thereof; and

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