WO2022088400A1 - Procédé de fabrication de variation de structure chromosomique - Google Patents

Procédé de fabrication de variation de structure chromosomique Download PDF

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WO2022088400A1
WO2022088400A1 PCT/CN2020/134673 CN2020134673W WO2022088400A1 WO 2022088400 A1 WO2022088400 A1 WO 2022088400A1 CN 2020134673 W CN2020134673 W CN 2020134673W WO 2022088400 A1 WO2022088400 A1 WO 2022088400A1
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dna
cells
preset
protein kinase
expression vector
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戴俊彪
温栾
陈佩双
卢俊南
林鑫
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1024—In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application belongs to the technical field of gene editing, and specifically relates to a method for producing chromosome structural variation.
  • 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
  • the internal factors include metabolic disorders and aging in organisms.
  • the main types are deletion, duplication, inversion and translocation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-1 ⁇ M; 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 ⁇ M-10 ⁇ M; 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.
  • the forming a predetermined chromosome break comprises: constructing a CRISPR system for the predetermined chromosome break; and introducing the CRISPR system into a cell.
  • the construction of a CRISPR system for preset chromosome breakage includes: synthesizing a pCS2-Cas9 expression vector using a preset primer sequence; vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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'-TCAAGAAAT
  • DNA-PK is a key protein kinase in the non-homologous recombination end repair (NHEJ) pathway in DNA damage repair.
  • NHEJ non-homologous recombination end repair
  • 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.
  • FIG. 1 is a schematic flow chart of an embodiment of a method for producing chromosomal structural variation of the present application
  • FIG. 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
  • FIG. 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.
  • ZFN zinc finger nucleases
  • TALEN transcription activator-like effector nuclease technology
  • CRISPR clustered regularly interspaced short palindromic repeats
  • 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.
  • DNA-dependent protein kinase is a key protein kinase in the non-homologous recombination end repair (NHEJ) pathway in DNA damage repair.
  • NHEJ non-homologous recombination end 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.
  • FIG. 1 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:
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the formation of preset chromosomal breaks includes:
  • Construction of a CRISPR system for pre-set chromosomal breaks can be accomplished in at least two ways:
  • the first method A-1 is a first method A-1:
  • 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 expression vector 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 is a first method A-2:
  • the target template DNA is transcribed using RNA transcription reagents and purified to obtain sgRNA.
  • 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.
  • 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:
  • the first method B-1 needs to be correspondingly used to introduce the CRISPR system into the cell:
  • the first method B-1 is a 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.
  • step S101 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:
  • RNP nucleic acid protein complex
  • 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.
  • 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.
  • DNA-PK DNA-dependent protein kinase
  • 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.
  • 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.
  • 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.
  • DNA-PK DNA-dependent protein kinase
  • 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.
  • 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:
  • 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:
  • NPM 1-sg F 5'-ACCGTGAACCCAGTAGCAGTTCG-3';
  • NPM1-sg R 5'-AAACCGAACTGCTACTGGGTTCAC-3'.
  • ALK is located on chromosome 2 of the human genome:
  • ALK-sg F 5'-ACCGTCGGTCCATTGCATAGAGG-3';
  • 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.
  • the transfection time can be adjusted in real time according to the actual transfection conditions and transfection progress.
  • 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;
  • DNA-dependent protein kinase inhibitor M3814 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.
  • the parameters are selected as above.
  • the first preset concentration is 100nM-1 ⁇ M, such as 100nM, 500nM or 1 ⁇ M, 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.
  • 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.
  • 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 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.
  • Example 2 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:
  • 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:
  • EWSR1-sg F 5'-ACCGGGGCATCCAAGATGTTAGC-3';
  • EWSR1-sg R 5'-AAACGCTAACATCTTGGATGCCCC-3'.
  • WT1 is located on chromosome 11 of the human genome:
  • WT1-sg F 5'-ACCGGGCTGAGCCCTTTATGTGA-3';
  • WT1-sg R 5'-AAACTCACATAAAGGGCTCAGCCC-3'.
  • 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;
  • DNA-dependent protein kinase inhibitor M3814 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.
  • the parameters are selected as above.
  • the first preset concentration is 100nM-1 ⁇ M, such as 100nM, 500nM or 1 ⁇ M, 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.
  • 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'.
  • 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.
  • breaks are formed in the two chromosomes under CRISPR cleavage.
  • 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.
  • CRISPR targets these two chromosomes to cut and reconnect to form a WT1-EWSR1 chromosomal translocation.
  • Example 1 and Example 2 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.
  • the DNA-PK inhibitor can be used to temporarily inhibit DNA-PK activity, which can significantly improve the efficiency of chromosomal translocation.
  • 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:
  • the default chromosomes are human genome chromosome 5 and human genome chromosome 2.
  • 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:
  • the preset translocation site ALK is located on chromosome 2 of the human genome:
  • 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).
  • RNA transcription reagent provided by the kit to transcribe the target template DNA to obtain sgRNA, and purify to obtain sgRNA.
  • 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.
  • sgRNA and 1.6 ⁇ g Cas9 protein were mixed and incubated at room temperature for 10 minutes to obtain nucleic acid-protein complexes; then mixed with 20 ⁇ L of electroporation solution containing 3.0 ⁇ 10 5 cells, transferred to a 20 ⁇ L 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.
  • DNA-dependent protein kinase inhibitor M3814 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.
  • the parameters are selected as above.
  • the first preset concentration is 100nM-1 ⁇ M, such as 100nM, 500nM or 1 ⁇ M, 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.
  • 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.
  • 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 A-2 combined with B-2 method was used to construct a CRISPR system for preset chromosomal translocation and introduce it into cells, specifically:
  • 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. .
  • the method of introducing the CRISPR system into cells is the same as that in Example 3, and will not be repeated here.
  • 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.
  • the parameters are selected as above.
  • the first preset concentration is 1 ⁇ M-10 ⁇ M, such as 1 ⁇ M, 5 ⁇ M or 10 ⁇ M, 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.
  • 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.
  • Example 3 and Example 4 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.
  • breaks are formed in the two chromosomes under CRISPR cleavage.
  • an NPM-ALK translocation is formed, which can be amplified by the primers indicated by the black arrows shown on the right. target fragment.
  • Example 3 and Example 4 The specific detection results of Example 3 and Example 4 are shown in Figure 4.
  • 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.

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Abstract

L'invention concerne un procédé de production de variation chromosomique. Le procédé comprend les étapes consistant à : former une rupture de chromosome prédéfinie ; et traiter des cellules à l'aide d'un inhibiteur de protéine kinase dépendant de l'ADN, de manière à obtenir un chromosome prédéfini après une variation structurelle. L'ADN-PK est une protéine kinase clé dans une voie de réparation d'extrémité de recombinaison non homologue dans la réparation d'endommagement de l'ADN. Lorsqu'un système CRISPR est utilisé pour provoquer des extrémités brisées d'ADN, l'inhibiteur de protéine kinase dépendant de l'ADN est utilisé pour inhiber temporairement l'activité de l'ADN-PK, de telle sorte que les extrémités d'ADN générées ne peuvent pas être rapidement connectées par un système de recombinaison non homologue de réparation d'endommagement d'ADN in vivo, l'opportunité d'un contact d'extrémité cassé d'ADN entre les chromosomes est augmentée et la probabilité de variation de structure chromosomique est augmentée. Le procédé est utilisé pour construire un modèle de maladie de variation de structure chromosomique.
PCT/CN2020/134673 2020-11-02 2020-12-08 Procédé de fabrication de variation de structure chromosomique WO2022088400A1 (fr)

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CN115948477A (zh) * 2022-07-20 2023-04-11 东北农业大学 一种提高CRISPR/Cas9的同源重组修复效率的诱导剂、方法以及应用

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