WO2022088400A1 - Method for making chromosome structure variation - Google Patents

Abstract

Provided is a method for making a chromosome structure variation. The method comprises: forming preset chromosome breakage; and treating cells by using a DNA dependent protein kinase inhibitor, so as to obtain a preset chromosome after structural variation. DNA-PK is a key protein kinase in a non-homologous recombination terminal repair (NHEJ) pathway in DNA damage repair. When a CRISPR system is used for causing DNA broken ends, the DNA dependent protein kinase inhibitor is used for temporarily inhibiting the activity of DNA-PK, so that the generated DNA ends cannot be quickly connected by an in-vivo DNA damage repair NHEJ system, the opportunity for DNA broken end contact between chromosomes is increased, and the probability of chromosome structure variation is increased. The method is used to construct a disease model of chromosome structure variation.

Description

A method for creating structural variation in chromosomes 【Technical field】

The present application belongs to the technical field of gene editing, and specifically relates to a method for producing chromosome structural variation.

【Background technique】

Chromosomal structural variation is a kind of chromosomal variation, which is the result of the combined action of internal and external factors. The external factors include various rays, chemical agents, and drastic changes in temperature, and the internal factors include metabolic disorders and aging in organisms. The main types are deletion, duplication, inversion and translocation. Among them, taking chromosomal translocation as an example, chromosomal translocation is the change of the chromosomal location segment, including the location segment change within a single chromosome and the location segment change between chromosomes. Chromosomal translocations can be further divided into non-reciprocal translocations and reciprocal translocations. The occurrence of chromosomal translocation requires two conditions, one is the presence of DNA breaks on both chromosomes at the same time; the other is the DNA damage repair mechanism in vivo to reconnect the DNA ends between different chromosomes.

Existing DNA breaks on chromosomes in cell lines usually use gene editing systems to target chromosomes separately, thereby causing DNA double-strand breaks. The broken DNA ends are randomly reconnected during the in vivo repair process. When the DNA ends are reconnected, there is a certain possibility of reconnecting the broken ends of the non-original chromosomal segments, resulting in chromosomal structural variation. However, the in vivo DNA repair system is more likely to reconnect the two DNA ends generated by the same DNA molecule damage. Such repair does not produce chromosome structural variation, and the dislocation connection between chromosomes completely depends on the probability of random connection. Therefore, this method makes chromosomes Structural variation is inefficient.

[Content of the invention]

The present application provides a method for producing chromosomal structural variation, so as to solve the technical problem of low efficiency of chromosomal structural variation.

In order to solve the above-mentioned technical problem, a technical solution adopted in the present application is: a method for producing a chromosomal translocation, comprising: forming a preset chromosomal break; treating the cell with a DNA-dependent protein kinase inhibitor to obtain a structurally mutated Preset chromosomes.

According to an embodiment of the present application, the treating the cells with a DNA-dependent protein kinase inhibitor includes: treating the cells with a DNA-dependent protein kinase inhibitor at a first preset concentration for a first preset time; stopping all The effect of the DNA-dependent protein kinase inhibitor on the cells is further cultured for a second preset time, and the cells are collected.

According to an embodiment of the present application, the treating the cells with a DNA-dependent protein kinase inhibitor includes: culturing the cells with a medium containing a DNA-dependent protein kinase inhibitor M3814 for 10h-20h, the DNA-dependent protein kinase inhibitor M3814 The first preset concentration of protein kinase inhibitor M3814 is 100nM-1μM; replace the medium without the DNA-dependent protein kinase inhibitor M3814 to culture the cells for 24h-32h, and collect the cells; The cells were cultured for 10h-20h in the medium of DNA-dependent protein kinase inhibitor KU57788, and the first preset concentration of the DNA-dependent protein kinase inhibitor KU57788 was 1 μM-10 μM; it was changed to not contain the DNA-dependent protein kinase The cells were cultured for 24h-32h in medium with kinase inhibitor M3814, and the cells were harvested.

According to an embodiment of the present application, the forming a predetermined chromosome break comprises: constructing a CRISPR system for the predetermined chromosome break; and introducing the CRISPR system into a cell.

According to an embodiment of the present application, the construction of a CRISPR system for preset chromosome breakage includes: synthesizing a pCS2-Cas9 expression vector using a preset primer sequence; vector.

According to an embodiment of the present application, the introduction of the CRISPR system into a cell includes: using liposomes to mix the pCS2-Cas9 expression vector and the sgRNA expression vector to transfect the cells for a third preset time, so that the cells are transfected for a third preset time. The pCS2-Cas9 expression vector and the sgRNA expression vector are introduced into the cells.

According to an embodiment of the present application, the construction of a CRISPR system for presetting chromosome breakage further includes: sequencing the pCS2-Cas9 expression vector and the sgRNA expression vector to verify the pCS2-Cas9 expression vector, and using the pCS2-Cas9- The cells were transfected with the Cas9 expression vector and the sgRNA expression vector.

According to an embodiment of the present application, the construction of a CRISPR system for preset chromosome breakage includes: synthesizing a forward primer and a reverse primer of the target template DNA; using the forward primer and reverse primer of the target template DNA, through Amplify the target template DNA by polymerase chain reaction; use RNA transcription reagent to transcribe the target template DNA, and purify to obtain sgRNA; obtain the Cas9 wild-type protein expression vector, and transform the strain to induce the Cas9 wild-type protein expression vector Expressed in the strain; harvested and disrupted the strain, harvested and purified the Cas9 protein.

According to an embodiment of the present application, the introduction of the CRISPR system into a cell includes: mixing and incubating the sgRNA and the Cas9 protein at room temperature to obtain a nucleic acid-protein complex; mixing the nucleic acid-protein complex with the The electroporation solution of the cells is mixed, and the cells are electroporated to introduce the nucleic acid-protein complex into the cells.

According to an embodiment of the present application, the method includes: synthesizing a forward primer and a reverse primer of the structurally mutated preset chromosomal gene; using the forward primer and the reverse primer to perform polymerase chain reaction , to amplify the preset chromosomal gene after the structural variation; observe the translocation effect of the preset chromosome by electrophoresis analysis; the sequence of the forward primer is selected from: SEQ ID NO1:5'-CAGTTGCTTGGTTCCCAGTT-3 '; and SEQ ID NO2:5'-GGGGAGAGGAAATCTTGCTG-3'; or as: SEQ ID NO3:5'-GTTGCTCACTTCTCTTGGGGCT-3'; the sequence of the reverse primer is selected from as: SEQ ID NO4:5'-AGGAATTGGCCTGCCTTAGT-3 '; and SEQ ID NO5:5'-GCAGCTTCAGTGCAATCACA-3'; or as: SEQ ID NO6:5'-TCAAGAAATGAAAACAGAGCCAGGT-3'.

The beneficial effects of the present application are: DNA-PK is a key protein kinase in the non-homologous recombination end repair (NHEJ) pathway in DNA damage repair. In the present application, when the CRISPR system is used to cause DNA breakage ends, the DNA-PK activity is temporarily inhibited by using a DNA-dependent protein kinase inhibitor, so that the generated DNA ends cannot be rapidly connected by the in vivo DNA damage repair NHEJ system, thus increasing the amount of interchromosomal DNA. The chance of breaking end contacts, thereby increasing the probability of chromosomal structural variation, is used in the construction of chromosomal structural variation disease models.

【Description of drawings】

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the drawings that are used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, under the premise of no creative work, other drawings can also be obtained from these drawings, wherein:

FIG. 1 is a schematic flow chart of an embodiment of a method for producing chromosomal structural variation of the present application;

2 is a schematic diagram of a chromosomal translocation in a specific embodiment of the method for producing chromosomal structural variation of the present application;

3 is a schematic diagram of the effect of detecting translocations in specific embodiment 1 and specific embodiment 2 in the method for producing chromosomal structural variation of the present application;

FIG. 4 is a schematic diagram showing the effect of detecting translocations in specific embodiment 3 and specific embodiment 4 in the method for producing chromosomal structural variation of the present application.

【Detailed ways】

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

To achieve chromosomal structural variation in cell lines, it is usually first to use zinc finger nucleases (ZFN), transcription activator-like effector nuclease technology (TALEN) or CRISPR (clustered regularly interspaced short palindromic repeats) gene editing systems, respectively, to target chromosomes, thereby Causes DNA double-strand breaks, and the broken DNA ends are randomly reconnected during repair in vivo. When the DNA ends are reconnected, there is a certain possibility that the broken ends of the non-original connected chromosomal fragments will be reconnected, resulting in chromosomal structural variation.

The applicant's research has found that DNA damage can be repaired in various ways. The homologous recombination repair pathway depends on the repair template, and the DNA damage site is repaired according to the sequence of the repair template; non-homologous recombination DNA end joining (NHEJ) does not depend on the repair template. , the broken DNA ends can be rejoined. Non-homologous recombination DNA end joining is also divided into two pathways, one is the canonical pathway (canonical NHEJ), which depends on XRCC4 and LIG4 and PARP3; Source end joining (MMHJ), dependent on CtIP and PARP1. Taking chromosomal translocations in chromosomal structural variation as an example, chromosomal translocations are mainly through the classic non-homologous recombination DNA end joining pathway. When this pathway is blocked, MMEJ will also play a role to generate chromosomal translocations. DNA-dependent protein kinase (DNA-PK) is a key protein kinase in the non-homologous recombination end repair (NHEJ) pathway in DNA damage repair. Inhibition of DNA-PK generally enhances the effect of radiation on cancer cells because inhibition of DNA-PK reduces the ability of cancer cells to repair the genome, thereby activating apoptotic pathways. Inhibition of DNA-PK can reduce the frequency of NHEJ, which can be used to increase the efficiency of homologous recombination. Therefore, the present application achieves an increase in the probability of chromosomal structural variation by inhibiting DNA-PK.

Specifically, please refer to FIG. 1 , which is a schematic flowchart of an embodiment of a method for producing a chromosomal structural variation of the present application.

The application provides a method for making chromosome structural variation, comprising the following steps:

S11: Formation of a preset chromosomal break.

To achieve preset chromosome breaks, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR (clustered regularly interspaced short palindromic repeats) gene editing systems are usually used to target preset chromosomes, thereby causing DNA damage. double-strand breaks.

Taking the CRISPR gene editing system as an example, the formation of preset chromosomal breaks includes:

One: Construction of a CRISPR system for preset chromosomal breaks.

Construction of a CRISPR system for pre-set chromosomal breaks can be accomplished in at least two ways:

The first method A-1:

1. Use the preset primer sequences to synthesize the pCS2-Cas9 expression vector. The preset primer sequences are shown in Table 1 below:

Table 1 Primer sequences

The primers in Table 1 were synthesized and PCR amplified with Q5 high-fidelity enzyme, and the pCS2-Cas9 expression vector was constructed by seamless cloning technology.

2. Separately synthesize sgRNA expression vectors related to predetermined chromosomal breakage sites, and sgRNA targeting sites related to predetermined chromosomal translocation sites can be obtained by consulting references.

In order to ensure the successful construction of the expression vector, it can be verified by sequencing whether the sequences of the pCS2-Cas9 expression vector and sgRNA expression vector are the same as the preset ones.

The second method A-2:

Using the forward primer and reverse primer of the target template DNA, amplify the target template DNA by polymerase chain reaction;

The target template DNA is transcribed using RNA transcription reagents and purified to obtain sgRNA.

Obtain the Cas9 wild-type protein expression vector, transform the strain, and induce the Cas9 wild-type protein expression vector to be expressed in the strain;

The strains were collected and disrupted, and the Cas9 protein was collected and purified.

A CRISPR system for preset chromosome breakage can be constructed by both methods. Of course, in other embodiments, other methods can also be used to construct the CRISPR system.

Two: Introducing the CRISPR system into cells.

The introduction of the CRISPR system into cells can also be achieved by at least the following two methods of B-1 and B-2:

Wherein, if the first method A-1 in step S101 is used to construct the CRISPR system for preset chromosome breakage, the first method B-1 needs to be correspondingly used to introduce the CRISPR system into the cell:

The first method B-1:

The pCS2-Cas9 expression vector and the sgRNA expression vector were mixed with liposomes to transfect the cells for a third preset time, so as to introduce the pCS2-Cas9 expression vector and the sgRNA expression vector into the cells.

If the second method A-2 in step S101 is used to construct the CRISPR system for preset chromosome breakage, the second method B-2 needs to be used to introduce the CRISPR system into the cell:

Second method B-2:

The sgRNA and Cas9 protein were mixed and incubated at room temperature to obtain a nucleic acid protein complex (RNP);

The nucleic acid protein complex is mixed with the electroporation solution containing the cells, and the cells are electroporated to introduce the nucleic acid protein complex into the cells.

Through the above two methods, the CRISPR system can be introduced into cells to target preset chromosomes, thereby causing DNA double-strand breaks.

S12: Treatment of cells with DNA-dependent protein kinase inhibitors to obtain pre-set chromosomes after structural variation.

Cells are treated with DNA-dependent protein kinase (DNA-PK) inhibitors, including:

Treat the cells with a first preset concentration of a DNA-dependent protein kinase inhibitor for a first preset time;

The effect of the DNA-dependent protein kinase inhibitor on the cells is stopped, the culture is continued for a second preset time, the cells are collected, and the preset chromosomes in the cells undergo structural variation.

Among them, the DNA-dependent protein kinase inhibitor can be selected from DNA-dependent protein kinase inhibitor M3814 or DNA-dependent protein kinase inhibitor KU57788, and in other embodiments, other DNA-dependent protein kinase inhibitors can also be selected.

It should be noted that, the first preset time, the second preset time, etc. can be specifically adjusted according to the type of DNA-dependent protein kinase (DNA-PK) inhibitor and the first preset concentration.

In the present application, when the CRISPR system is used to cause DNA breakage ends, the DNA-PK activity is temporarily inhibited by using a DNA-dependent protein kinase inhibitor, so that the generated DNA ends cannot be rapidly connected by the in vivo DNA damage repair NHEJ system, thus increasing the amount of interchromosomal DNA. The chance of breaking end contacts, thereby increasing the probability of chromosomal structural variation, is used in the construction of chromosomal structural variation disease models.

Described below through several specific embodiments:

Example 1:

Example 1 A-1 combined with B-1 method was used to construct a CRISPR system for preset chromosomal translocation, and introduce it into cells, specifically:

1. Construction of a CRISPR system for preset chromosomal translocations,

The pCS2-Cas9 expression vector was synthesized using preset primer sequences. The preset primer sequences are shown in Table 1 above.

The primers in Table 1 were synthesized and PCR amplified with Q5 high-fidelity enzyme, and the pCS2-Cas9 expression vector was constructed by seamless cloning technology.

The preset chromosomes are chromosome 5 of the human genome and chromosome 2 of the human genome, and sgRNA expression vectors related to the translocation sites of the preset chromosomes are synthesized respectively. The specific sgRNA targeting sites related to the translocation sites of the preset chromosomes are as follows :

NPM is located on chromosome 5 of the human genome:

SEQ ID NO 11:

NPM 1-sg F: 5'-ACCGTGAACCCAGTAGCAGTTCG-3';

SEQ ID NO 12:

NPM1-sg R: 5'-AAACCGAACTGCTACTGGGTTCAC-3'.

ALK is located on chromosome 2 of the human genome:

SEQ ID NO 13:

ALK-sg F: 5'-ACCGTCGGTCCATTGCATAGAGG-3';

SEQ ID NO 14:

ALK-sg R: 5'-AAACCCTCTATGCAATGGACCGAC-3'.

The same amount of F and R oligonucleotides were mixed, denatured at high temperature, annealed to form short double-stranded DNA, and the short double-stranded DNA formed by annealing was ligated to the restriction endonuclease Bsa I-treated pGL3- On the U6-sgRNA vector, the sgRNA expression vector related to the translocation site of the preset chromosome is formed. The pCS2-Cas9 expression vector and sgRNA expression vector were verified by sequencing.

2. Introduce the CRISPR system into cells.

One day before transfection, 5×10 ⁵ 293T cells were seeded into one well of a 24-well plate, and the next day, 250ng of pCS2-Cas9 expression vector and 125ng of sgRNA expression vector pGL3-U6-sgRNA-ALK were mixed with Fugene liposomes. , and 125ng of pGL3-U6-sgRNA-NPM, and the mixture was used to transfect the cells for 6h to introduce the pCS2-Cas9 expression vector and the sgRNA expression vector into the cells, and the cells were human embryonic kidney cells. Of course, in other embodiments, the transfection time can be adjusted in real time according to the actual transfection conditions and transfection progress.

3. Treat cells with DNA-dependent protein kinase inhibitors to obtain pre-translocated chromosomes.

The cells were treated with DNA-dependent protein kinase inhibitor M3814 at a first preset concentration of 500nM for a first preset time of 18h;

The effect of DNA-dependent protein kinase inhibitor M3814 on the cells was stopped, and the cells were cultured for a second preset time of 30 h by replacing with a medium without DNA-dependent protein kinase inhibitor M3814, and the cells were collected.

In this embodiment, the parameters are selected as above. During the experimental operation, the first preset concentration is 100nM-1μM, such as 100nM, 500nM or 1μM, etc. The first preset concentration should not be too high, which will cause toxicity to cells. The first preset time is 10h-20h, for example, 10h, 14h, 18h, or 20h. The second preset time is 24h-32h, for example, 24h, 28h, 30h, or 32h.

4. Detect the translocation effect of preset chromosomes.

Synthesize the forward primer and reverse primer of the preset chromosomal gene after translocation;

Use forward primer and reverse primer to carry out polymerase chain reaction to amplify the preset chromosomal gene after translocation;

The translocation effect of preset chromosomes was observed by electrophoresis analysis.

The NPM-ALK chromosomal translocation utilizes two sets of primers, the first set: NPM-ALK-F1 (SEQ ID NO1): 5'-CAGTTGCTTGGTTCCCAGTT-3' and NPM-ALK-R1 (SEQ ID NO4): 5'-AGGAATTGGCCTGCCTTAGT- 3'; the second group: NPM-ALK-F2 (SEQ ID NO2): 5'-GGGGAGAGGAAATCTTGCTG-3' and NPM-ALK-R2 (SEQ ID NO5): 5'-GCAGCTTCAGTGCAATCACA-3' were detected by nested PCR.

The advantage of nested PCR is that if the first amplification produces an erroneous fragment, the probability of primer pairing and amplification on the erroneous fragment the second time is extremely low. Therefore, the amplification of nested PCR is very specific and the detection accuracy is high.

Example 2:

In Example 2, the A-1 combined with B-1 method was also used to construct a CRISPR system for preset chromosomal translocation and introduce it into cells.

1. Construction of a CRISPR system for preset chromosomal translocations.

The method for constructing the CRISPR system for preset chromosomal translocation in Example 2 is basically the same as that in Example 1, which will not be repeated here, and the differences are:

In Example 2, the preset chromosomes are human genome chromosome 22 and human genome chromosome 11, and the specific sgRNA targeting sites related to the translocation sites of the preset chromosomes are as follows:

EWSR1 is located on chromosome 22 of the human genome:

SEQ ID NO 15:

EWSR1-sg F: 5'-ACCGGGGCATCCAAGATGTTAGC-3';

SEQ ID NO 16:

EWSR1-sg R: 5'-AAACGCTAACATCTTGGATGCCCC-3'.

WT1 is located on chromosome 11 of the human genome:

SEQ ID NO 17:

WT1-sg F: 5'-ACCGGGCTGAGCCCTTTATGTGA-3';

SEQ ID NO 18:

WT1-sg R: 5'-AAACTCACATAAAGGGCTCAGCCC-3'.

2. Introduce the CRISPR system into cells.

One day before transfection, 5×10 ⁵ 293T cells were seeded into one well of a 24-well plate, and the next day, 250ng of pCS2-Cas9 expression vector and 125ng of sgRNA expression vector pGL3-U6-sgRNA-WT1 were mixed with Fugene liposomes. , and 125ng of pGL3-U6-sgRNA-EWSR1, and the mixture was used to transfect the cells, and the third preset time was 6h to introduce the pCS2-Cas9 expression vector and the sgRNA expression vector into the cells, and the cells were human embryonic kidney cells. Of course, the third preset time can be adjusted in real time according to actual transfection conditions and transfection progress.

3. Treat cells with DNA-dependent protein kinase inhibitors to obtain pre-translocated chromosomes.

The cells were treated with DNA-dependent protein kinase inhibitor M3814 at a first preset concentration of 500nM for a first preset time of 18h;

The effect of DNA-dependent protein kinase inhibitor M3814 on cells was stopped, and the cells were cultured for a second preset time of 30 h by replacing with a medium without DNA-dependent protein kinase inhibitor M3814, and cells were collected.

In this embodiment, the parameters are selected as above. During the experimental operation, the first preset concentration is 100nM-1μM, such as 100nM, 500nM or 1μM, etc. The first preset concentration should not be too high, which will cause toxicity to cells. The first preset time is 10h-20h, for example, 10h, 14h, 18h, or 20h. The second preset time is 24h-32h, for example, 24h, 28h, 30h, or 32h.

4. Detect the translocation effect of preset chromosomes.

Synthesize the forward primer and reverse primer of the preset chromosomal gene after translocation;

Use forward primer and reverse primer to carry out polymerase chain reaction to amplify the preset chromosomal gene after translocation;

The translocation effect of preset chromosomes was observed by electrophoresis analysis.

EWSR-WT translocation was detected by PCR using primers as follows: EWSR-WT-F (SEQ ID NO 3): 5'-GTTGCTCACTTCTCTTGGGGCT-3' and EWSR-WT-R (SEQ ID NO 6): 5'-TCAAGAAATGAAAACAGAGCCAGGT-3'.

Analysis: The chromosomal translocation effects of Examples 1 and 2 are shown in FIG. 2 . The NPM and ALK gene loci are located on chromosome 5 and chromosome 2 of the human genome, respectively, and the arrows below or above the gene name indicate the direction of transcription of the gene. As shown in the upper left of Figure 2, breaks are formed in the two chromosomes under CRISPR cleavage. When a chromosomal translocation occurs, an NPM-ALK translocation is formed, which can be amplified by the primers indicated by the black arrows shown on the right. target fragment. EWSR1 and WT1 are located on chromosome 22 and chromosome 11 of the human genome, respectively. As shown in the lower left of Figure 2, CRISPR targets these two chromosomes to cut and reconnect to form a WT1-EWSR1 chromosomal translocation.

The specific detection results of Example 1 and Example 2 are shown in FIG. 3 . The DNA-PK inhibitor M3814 significantly increased NPM-ALK and WT1-EWSR1 chromosomal translocations in human embryonic kidney cells. The CRISPR system targeting NPM and ALK gene loci, or the CRISPR system targeting WT1 and EWSR1 gene loci were introduced into human embryonic kidney 293T cells, and then the cells were treated with DNA-PK inhibitor M3814, and the cells were collected to detect chromosomal translocation. bit effect. WT represents cells without the introduction of the CRISPR system, and no chromosomal translocations were detected. Chromosomal translocations were detected in all cells introduced with the CRISPR system, and significantly stronger chromosomal translocation signals were found in M3814-treated cells. Actin beta is an internal reference gene.

From the above results, it can be seen that on the basis of introducing CRISPR to cut different chromosomes to cause chromosomal double-strand breaks, the DNA-PK inhibitor can be used to temporarily inhibit DNA-PK activity, which can significantly improve the efficiency of chromosomal translocation.

Example 3:

Example 3 A-2 combined with B-2 method was used to construct a CRISPR system for preset chromosomal translocation and introduce it into cells, specifically:

1. Construction of a CRISPR system for preset chromosomal translocations.

The default chromosomes are human genome chromosome 5 and human genome chromosome 2.

Synthesize the forward primer and reverse primer of the target template DNA. The target template DNA is used to transcribe the sgRNA. The forward primer and reverse primer of the target template DNA are as follows:

The preset translocation site NPM is located on chromosome 5 of the human genome:

SEQ ID NO 19: NPM-F:

5'-TAATACGACTCACTATAGTGAACCCAGTAGCAGTTCG-3';

SEQ ID NO20: NPM-R:

5'-TTCTAGCTCTAAAACCGAACTGCTACTGGGTTCA-3';

The preset translocation site ALK is located on chromosome 2 of the human genome:

SEQ ID NO21: ALK-F:

5'-TAATACGACTCACTATAGTCGGTCCATTGCATAGAG-3';

SEQ ID NO22: ALK-R:

5'-TTCTAGCTCTAAAACCCTCTATGCAATGGACCGAC-3'.

Using the forward primer and reverse primer of the target template DNA, the target template DNA is amplified by polymerase chain reaction according to the conditions provided by the kit. The kit can be selected from the Invitrogen precision gRNA synthesis kit (Cat. No. A29377).

Use the RNA transcription reagent provided by the kit to transcribe the target template DNA to obtain sgRNA, and purify to obtain sgRNA.

Obtain the Cas9 wild-type protein expression vector (the Cas9 wild-type protein expression vector was purchased from Addgene (Cat. No. 62374)), transform the strain, and induce the Cas9 wild-type protein expression vector to express in the strain overnight at 20°C and 0.5 mM IPTG.

Bacterial cells were collected the next day, strains were sonicated, and Cas9 protein was affinity-purified with Ni-NTA, followed by ion-exchange purification of Cas9 protein on an AKTA instrument.

2. Introduce the CRISPR system into cells.

0.55ug sgRNA and 1.6μg Cas9 protein were mixed and incubated at room temperature for 10 minutes to obtain nucleic acid-protein complexes; then mixed with 20μL of electroporation solution containing 3.0×10 ⁵ cells, transferred to a 20μL electroporation tube, and passed on an electroporator 380V, 30ms parameters electroporation cells, so as to introduce the nucleic acid protein complex into the cells, the cells are human embryonic kidney cells.

3. Treat cells with DNA-dependent protein kinase inhibitors to obtain pre-translocated chromosomes.

Immediately after electroporation, cells were treated with DNA-dependent protein kinase inhibitor M3814 at a first preset concentration of 500nM for a first preset time of 16h;

The effect of DNA-dependent protein kinase inhibitor M3814 on the cells was stopped, and the culture medium without DNA-dependent protein kinase inhibitor M3814 was replaced to culture the cells for a second preset time of 32 h, and then the cells were collected.

In this embodiment, the parameters are selected as above. During the experimental operation, the first preset concentration is 100nM-1μM, such as 100nM, 500nM or 1μM, etc. The first preset concentration should not be too high, which will cause toxicity to cells. The first preset time is 10h-20h, for example, 10h, 14h, 18h, or 20h. The second preset time is 24h-32h, for example, 24h, 28h, 30h, or 32h.

4. Detect the translocation effect of preset chromosomes.

Synthesize the forward primer and reverse primer of the preset chromosomal gene after translocation;

Use forward primer and reverse primer to carry out polymerase chain reaction to amplify the preset chromosomal gene after translocation;

The translocation effect of preset chromosomes was observed by electrophoresis analysis.

The NPM-ALK chromosomal translocation utilizes two sets of primers, the first set: NPM-ALK-F1 (SEQ ID NO1): 5'-CAGTTGCTTGGTTCCCAGTT-3' and NPM-ALK-R1 (SEQ ID NO4): 5'-AGGAATTGGCCTGCCTTAGT- 3'; the second group: NPM-ALK-F2 (SEQ ID NO2): 5'-GGGGAGAGGAAATCTTGCTG-3' and NPM-ALK-R2 (SEQ ID NO5): 5'-GCAGCTTCAGTGCAATCACA-3' were detected by nested PCR.

The advantage of nested PCR is that if the first amplification produces an erroneous fragment, the probability of primer pairing and amplification on the erroneous fragment the second time is extremely low. Therefore, the amplification of nested PCR is very specific and the detection accuracy is high.

Example 4:

Example 4 A-2 combined with B-2 method was used to construct a CRISPR system for preset chromosomal translocation and introduce it into cells, specifically:

1. Construction of a CRISPR system for preset chromosomal translocations.

The preset chromosomes are human genome chromosome 5 and human genome chromosome 2, and the preset translocation sites are NPM and ALK. The method of constructing a CRISPR system for preset chromosomal translocation is the same as that in Example 3, and will not be repeated here. .

2. Introduce the CRISPR system into cells.

The method of introducing the CRISPR system into cells is the same as that in Example 3, and will not be repeated here.

3. Treat cells with DNA-dependent protein kinase inhibitors to obtain pre-translocated chromosomes.

Immediately after electroporation, cells were treated with DNA-dependent protein kinase inhibitor KU57788 at a first preset concentration of 10 μM for a first preset time of 18h;

The effect of the DNA-dependent protein kinase inhibitor KU57788 on the cells was stopped, and the cells were cultured for a second preset time of 30 h by replacing the medium without the DNA-dependent protein kinase inhibitor KU57788, and the cells were collected.

In this embodiment, the parameters are selected as above. During the experimental operation, the first preset concentration is 1 μM-10 μM, such as 1 μM, 5 μM or 10 μM, etc. The first preset concentration should not be too high, which will cause toxicity to cells. The first preset time is 10h-20h, for example, 10h, 14h, 16h, 18h, or 20h. The second preset time is 24h-32h, for example, 24h, 28h, 30h, or 32h.

4. Detect the translocation effect of preset chromosomes.

The method for detecting the translocation effect of the preset chromosome is the same as that in Embodiment 3, and will not be repeated here.

Analysis: The chromosomal translocation effect of Example 3 and Example 4 is shown in FIG. 2 . The NPM and ALK gene loci are located on chromosome 5 and chromosome 2 of the human genome, respectively, and the arrows below or above the gene name indicate the direction of transcription of the gene. As shown in the upper left of Figure 2, breaks are formed in the two chromosomes under CRISPR cleavage. When a chromosomal translocation occurs, an NPM-ALK translocation is formed, which can be amplified by the primers indicated by the black arrows shown on the right. target fragment.

The specific detection results of Example 3 and Example 4 are shown in Figure 4. After the CRISPR system introduced human embryonic kidney cells in the form of nucleic acid protein complex RNP, they were treated with DNA-PK inhibitors M3814 (500nM) and KU57788 (10uM) for 16 48 hours after the introduction of the CRISPR system, the cells were collected to detect the effect of chromosomal translocation. WT represents untreated human embryonic kidney cells without any chromosomal translocations. Both DNA-PK inhibitors can significantly enhance the CRISPR-induced chromosomal translocation effect.

From the above results, it can be seen that on the basis of introducing CRISPR to cut different chromosomes respectively to cause chromosomal double-strand breaks, the transient inhibition of DNA-PK activity by DNA-PK inhibitors can significantly improve the efficiency of chromosomal translocation.

The above descriptions are only the embodiments of the present application, and are not intended to limit the scope of the patent of the present application. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present application, or directly or indirectly applied to other related technologies Fields are similarly included within the scope of patent protection of this application. .

Claims (10)

1. A method for producing chromosome structural variation, comprising:

   Formation of preset chromosomal breaks;

   The cells are treated with a DNA-dependent protein kinase inhibitor to obtain structurally mutated pre-set chromosomes.
2. The method of claim 1, wherein the treating the cells with a DNA-dependent protein kinase inhibitor comprises:

   treating the cells with a first predetermined concentration of a DNA-dependent protein kinase inhibitor for a first predetermined time;

   Stop the action of the DNA-dependent protein kinase inhibitor on the cells, continue to culture for a second preset time, and collect the cells.
3. The method of claim 2, wherein the treating the cells with a DNA-dependent protein kinase inhibitor comprises:

   The cells are cultured for 10h-20h in a medium containing a DNA-dependent protein kinase inhibitor M3814, and the first preset concentration of the DNA-dependent protein kinase inhibitor M3814 is 100nM-1μM;

   Replace the medium without the DNA-dependent protein kinase inhibitor M3814 to culture the cells for 24h-32h, and collect the cells; or,

   The cells are cultured for 10h-20h in a medium containing a DNA-dependent protein kinase inhibitor KU57788, and the first preset concentration of the DNA-dependent protein kinase inhibitor KU57788 is 1 μM-10 μM;

   The cells were cultured for 24h-32h by changing to a medium without the DNA-dependent protein kinase inhibitor M3814, and the cells were collected.
4. The method of claim 1, wherein the forming a predetermined chromosome break comprises:

   constructing a CRISPR system for said predetermined chromosomal break;

   The CRISPR system is introduced into cells.
5. The method according to claim 4, wherein the constructing a CRISPR system for presetting chromosome breaks comprises:

   Synthesize pCS2-Cas9 expression vector using preset primer sequences;

   The sgRNA expression vectors related to the breakpoints of the preset chromosomes are synthesized respectively.
6. The method of claim 5, wherein the introducing the CRISPR system into a cell comprises:

   The pCS2-Cas9 expression vector and the sgRNA expression vector are mixed with liposomes to transfect the cells for a third preset time, so as to introduce the pCS2-Cas9 expression vector and the sgRNA expression vector into the cells.
7. The method according to claim 6, wherein the constructing a CRISPR system for presetting chromosome breaks further comprises:

   The pCS2-Cas9 expression vector and the sgRNA expression vector are verified by sequencing, and the cells are transfected with the pCS2-Cas9 expression vector and the sgRNA expression vector verified by the sequencing.
8. The method according to claim 4, wherein the constructing a CRISPR system for presetting chromosome breaks comprises:

   Synthesize forward and reverse primers for target template DNA;

   Using the forward primer and reverse primer of the target template DNA, amplify the target template DNA by polymerase chain reaction;

   Use RNA transcription reagent to transcribe the target template DNA, and purify to obtain sgRNA;

   Obtain the Cas9 wild-type protein expression vector, transform the strain, and induce the Cas9 wild-type protein expression vector to be expressed in the strain;

   The strains were collected and disrupted, and the Cas9 protein was collected and purified.
9. The method of claim 8, wherein the introducing the CRISPR system into a cell comprises:

   mixing and incubating the sgRNA and the Cas9 protein at room temperature to obtain a nucleic acid-protein complex;

   The nucleic acid-protein complex is mixed with an electroporation solution containing the cells, and the cells are electroporated to introduce the nucleic acid-protein complex into the cells.
10. The method of claim 1, wherein the method comprises:

    synthesizing the forward primer and reverse primer of the predetermined chromosomal gene after the structural variation;

    Use the forward primer and the reverse primer to carry out polymerase chain reaction to amplify the predetermined chromosomal gene after the structural variation;

    Observing the translocation effect of the preset chromosome by electrophoresis analysis;

    The sequence of the forward primer is selected from, for example:

    SEQ ID NO 1: 5'-CAGTTGCTTGGTTCCCAGTT-3'; and

    SEQ ID NO2: 5'-GGGGAGAGGAAATCTTGCTG-3';

    or as in:

    SEQ ID NO3: 5'-GTTGCTCACTTCTCTTGGGGCT-3';

    The sequence of the reverse primer is selected from the group consisting of:

    SEQ ID NO4: 5'-AGGAATTGGCCTGCCTTAGT-3'; and

    SEQ ID NO5: 5'-GCAGCTTCAGTGCAATCACA-3';

    or as in:

    SEQ ID NO 6: 5'-TCAAGAAATGAAAACAGAGCCAGGT-3'.

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