CN105602993A - Mitochondrion-targeted gene editing system and method - Google Patents
Mitochondrion-targeted gene editing system and method Download PDFInfo
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- CN105602993A CN105602993A CN201610035146.9A CN201610035146A CN105602993A CN 105602993 A CN105602993 A CN 105602993A CN 201610035146 A CN201610035146 A CN 201610035146A CN 105602993 A CN105602993 A CN 105602993A
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Abstract
The invention relates to a mitochondrion-targeted gene editing system and method. The system comprises: a, mitochondrion-targeted DNA nuclease, b, mitochondrion-targeted sgRNA, and c, mitochondrion-targeted RNA repair template. The method comprises: using the system to position the mitochondrion-targeted DNA nuclease, the mitochondrion-targeted sgRNA and the mitochondrion-targeted RNA repair template to a mitochondrion. By making a DNA double-strand break (DSB) at a target or near the target with sequence-specific nuclease and through the RNA template, homologous recombination repairing at the target is achieved.
Description
[technical field]
The present invention relates to a kind of gene editing system and method, be specifically related to a kind of Mitochondrially targeted gene and compileCollect system and method.
[background technology]
The general principle of genome fixed point editor/modification is utilized spontaneous in target site district or is brought out at presentDNA double chain breach (double-strainbreaks, DSBs), DSBs repairs the DNA in active cellMechanism is carried out genomic transformation, such as the end in non-homogeneous district connects (NHEJ) or homologous recombination (HR)(accompanying drawing 1).
In cell, the probability of the spontaneous generation of optimistic estimate DSB is lower than 1/104, if pass through genetic engineeringAdopt the nucleases such as spCas9 and SaCas9 to bring out DSBs, efficiency can be increased to more than 10%, and has positionPoint specificity, can bring out DSBs so that ensuing gene repair process is sent out in the site of endogenous gene sudden changeRaw. After DNA repair mechanism in DSBs active cell, have two kinds of different repair mechanisms emulativeParticipate in the reparation of DSBs, a kind of is the end connection (Non-homologousend of non-homologous regionJoint-NHEJ), one is homologous recombination (Homologousrecombination-HR). Realize genomeFixed point accurately edit, need to be by means of homologous recombination repair mechanism. Therefore directly improve DSBs repair processThe frequency that middle homologous recombination occurs or the end that suppresses non-homogeneous district connect (NHEJ) and all contribute to improve geneThe efficiency of group fixed point editor/modification.
Human genome comprises nuclear genome and mitochondrial genomes two parts, nuclear genome and lineThe genomic sudden change of plastochondria all may cause human inheritance's disease. Current gene editing technology mainly for beNuclear genome, therefore develops the gene editing technology repair line plastochondria gene pairs for mitochondrial genomesThe treatment of human diseases has important meaning equally.
Realize the editor of chondriogen, need to be positioned in mitochondria specific nuclease. ForCRISPR-Cas9 system, the sgRNA that is responsible for guiding Cas9 proteinic genome location need to enter equallyEntering in mitochondria to allow CRISPR-Cas9 system play a role in mitochondria. Cas9 albumen and sgRNAMitochondria location can realize knocking out of chondriogen, but to realize the fixed point editor of chondriogen,Also need the mitochondrial recovery template of target.
Existing gene site-directed editor's principle is mainly homologous recombination repair mechanism, therefore needs homology to repair mouldPlate. Existing report points out, the template that can mediate homologous recombination repair can be double-stranded DNA, DNA widowNucleotide chain or RNA template. Double-stranded DNA and DNA oligonucleotide chain are difficult to realize mitochondria location,But can realize the mitochondria location of RNA template by means of Mitochondrially targeted RNA signal.
[summary of the invention]
The object of the invention is to realize by means of Mitochondrially targeted RNA signal the mitochondria of RNA templateLocation.
To achieve these goals, provide a kind of Mitochondrially targeted gene editing system, comprising:
A. Mitochondrially targeted DNA nuclease,
B. Mitochondrially targeted sgRNA,
C. Mitochondrially targeted RNA recovery template.
Described DNA nuclease is SpCas9, SaCas9, CPF1, ZFN, TALENs.
The construction method of Mitochondrially targeted DNA nuclease is: design coding COX8 gene string plastochondria locationThe complementary oligonucleotide chain of sequence, is connected into the expression vector of SpCas9 after annealing, obtain COX8MTS-SpCas9 fusion, promoter is CBh.
The method of the sgRNA that Mitochondrially targeted sgRNA builds is: the RNA that design comprises mitochondria locationSignal and complementary oligonucleotide chain for the sgRNA of people source ND4 gene, be connected into sgRNA's after annealingExpression vector, obtains mitoRNAsignal-ND4sgRNA fusion sequence, and promoter is U6.
The preparation method of Mitochondrially targeted RNA recovery template is: adopt the synthetic mode of DNA, by lineThe RNA signal of plastochondria location merges and obtains with the people source ND4 recovery template with HA sequence labelMitoRNAsignal-ND4-HA sequence, enzyme is cut to connect and is entered U6 expression vector
A Mitochondrially targeted gene editing method, adopts above-mentioned arbitrary gene editing system, by mitochondriaThe DNA nuclease of target, Mitochondrially targeted sgRNA and Mitochondrially targeted RNA Template Location are to lineIn plastochondria.
Adopt Lipofectamine3000 transfection reagent by fixed to DNA nuclease, the mitochondria of mitochondria locationThe sgRNA of position and the expression vector cotransfection of RNA recovery template enter 293 clones, 37 degree carbon dioxideIncubator is cultivated 48 hours. Extract cell genomic dna, adopt the specific forward primer of HA and Ren YuanThe specific reverse primer of ND4 carries out PCR.
The present invention utilize sequence-specific nuclease target site place or near DNA double chain breach of manufacture(DSBs), realize the homologous recombination repair at target site place by RNA template.
[brief description of the drawings]
Fig. 1 is the gene editing principle schematic of DSBs mediation;
Fig. 2 is chondriogen reparation-system schematic diagram;
Behave source ND4 homologous complementary template of Fig. 3 builds schematic diagram;
Fig. 4 is that ND4-HA gene editing detects design of primers schematic diagram,
Fig. 5 is the PCR proof diagram of ND4-HA,
Fig. 6 is ND4-HA sequence verification, sequence alignment figure;
Fig. 7 source MT-TK gene loci editor that behaves, homologous complementary template builds schematic diagram;
Fig. 8 source MT-TK gene loci editor that behaves, detects primer schematic diagram;
Fig. 9 source MT-TK gene loci editor that behaves, PCR proof diagram;
Figure 10 source MT-TK gene loci editor that behaves, sequence verification sequence alignment figure.
[detailed description of the invention]
Be described further for the present invention below in conjunction with embodiment and accompanying drawing, embodiment and accompanying drawing are only for separatingRelease explanation and be not intended to limit the scope of the invention.
The gene site-directed HA label of knocking in of embodiment 1-mitochondria ND4
1. transformation nuclease SpCas9, builds the mito-SpCas9 that mitochondria is located
Design coding COX8 gene string plastochondria positioning sequence (mitochondrial-targetingsequenceMTS) complementary oligonucleotide chain, is connected into the expression vector of SpCas9 after annealing, obtain COX8MTS-SpCas9 fusion, promoter is CBh.
2. transformation sgRNA, builds the sgRNA that mitochondria is located
The RNA signal that design comprises mitochondria location and the complementation widow of the sgRNA for people source ND4 geneNucleotide chain, is connected into the expression vector of sgRNA after annealing, obtain mitoRNAsignal-ND4sgRNAFusion sequence, promoter is U6.
3. design and synthesize the recovery template with the ND4 complementation of people source
Adopt DNA synthetic mode, by the RNA signal of mitochondria location with HA sequence labelThe ND4 recovery template fusion of people source obtains mitoRNAsignal-ND4-HA sequence, and enzyme is cut to connect and entered U6Expression vector (Fig. 3).
4. the chondriogen edit effect of the new system of rotaring redyeing 293 cell system checking
Adopt Lipofectamine3000 transfection reagent by step 1, the 2 and 3 expression vector cotransfections that buildEnter 293 clones, 37 degree CO2gas incubators are cultivated 48 hours. Extract cell genomic dna, adoptCarry out PCR with the specific forward primer of HA and the people source specific reverse primer of ND4, adopt people sourceThe specific forward primer of ND4 and the specific reverse primer of HA carry out PCR (Fig. 4-5), reclaim objectAfter fragment, confirm mitochondrial editor's feasibility (Fig. 6) through order-checking.
Embodiment 2-utilize homologous recombination to replace with mouse mitochondrial MT-TK gene people source MT-TK gene
1. transformation nuclease SpCas9, builds the mito-SpCas9 that mitochondria is located
Design coding COX8 gene string plastochondria positioning sequence (mitochondrial-targetingsequenceMTS) complementary oligonucleotide chain, is connected into the expression vector of SpCas9 after annealing, obtain COX8MTS-SpCas9 fusion, promoter is CBh.
2. transformation sgRNA, builds the sgRNA that mitochondria is located
The RNA signal that design comprises mitochondria location and the complementation of the sgRNA for people source MT-TK geneOligonucleotide chain, is connected into the expression vector of sgRNA after annealing, obtain mitoRNAsignal-MT-TKSgRNA fusion sequence, promoter is U6.
3. design and synthesize the genome editing template with the MT-TK complementation of people source
Adopt DNA synthetic mode, by the RNA signal of mitochondria location with HA sequence labelThe MT-TK genome editing template fusion of people source obtains mitoRNAsignal-MT-TK-replacement sequence,Be wherein the MT-TK gene in mouse source by people source MT-TK Gene Replacement, enzyme is cut to connect and is entered U6 expressionCarrier (Fig. 7).
4. the chondriogen edit effect of the new system of rotaring redyeing 293 cell system checking
Adopt Lipofectamine3000 transfection reagent by step 1, the 2 and 3 expression vector cotransfections that buildEnter 293 clones, 37 degree CO2gas incubators are cultivated 48 hours. Extract cell genomic dna, adoptClose on sequence-specific anti-with the forward primer of mouse MT-TK gene specific and people source MT-TK geneCarry out PCR to primer, adopt people source MT-TK gene to close on sequence-specific forward primer and mouseThe reverse primer of MT-TK gene specific carries out PCR (Fig. 8-9), after recovery object fragment, confirms through order-checkingMitochondrial editor's feasibility (Figure 10).
Details as Follows in invention, to use the SpCas9 expression vector establishment of mitochondria location: according to existing reportRoad, adopts the synthetic mode of DNA to obtain SpCas9 expression vector; According to existing report, synthetic Cox8Mitochondria positioning sequence, is connected in SpCas9 expression vector.
SpCas9 coded sequence, as shown in SEQIDNO.1.
COX8 mitochondria positioning sequence, as shown in SEQIDNO.2.
SgRNA builds: synthetic oligonucleotide chain is connected into the expression vector of U6 promoters driven after annealing;U6 promoter adopts the synthetic mode of DNA to obtain.
ND4sgRNA: as shown in SEQIDNO.3.
MT-TKsgRNA is as shown in SEQIDNO.4.
Recovery template adopts the synthetic mode of DNA to obtain.
ND4-HA template: as shown in SEQIDNO.5.
MT-TK replaces template: as shown in SEQIDNO.6.
Claims (7)
1. a Mitochondrially targeted gene editing system, it is characterized in that comprising:
A. Mitochondrially targeted DNA nuclease,
B. Mitochondrially targeted sgRNA,
C. Mitochondrially targeted RNA recovery template.
2. Mitochondrially targeted gene editing system as claimed in claim 1, is characterized in that described DNA nuclease is SpCas9, SaCas9, CPF1, ZFN, TALENs.
3. Mitochondrially targeted gene editing system as claimed in claim 1, the construction method that it is characterized in that Mitochondrially targeted DNA nuclease is: the complementary oligonucleotide chain of design coding COX8 gene string plastochondria positioning sequence, after annealing, be connected into the expression vector of SpCas9, obtain COX8MTS-SpCas9 fusion, promoter is CBh.
4. Mitochondrially targeted gene editing system as claimed in claim 1, the method that it is characterized in that the sgRNA of Mitochondrially targeted sgRNA structure is: the RNA signal that design comprises mitochondria location and the complementary oligonucleotide chain of the sgRNA for people source ND4 gene, after annealing, be connected into the expression vector of sgRNA, obtain mitoRNAsignal-ND4sgRNA fusion sequence, promoter is U6.
5. Mitochondrially targeted gene editing system as claimed in claim 1, the preparation method that it is characterized in that Mitochondrially targeted RNA recovery template is: adopt the synthetic mode of DNA, the RNA signal of mitochondria location is merged and obtains mitoRNAsignal-ND4-HA sequence with the people source ND4 recovery template with HA sequence label, and enzyme is cut to connect and is entered U6 expression vector.
6. a Mitochondrially targeted gene editing method, is characterized in that adopting the arbitrary described gene editing system of claim 1～5, by Mitochondrially targeted DNA nuclease, Mitochondrially targeted sgRNA and Mitochondrially targeted RNA Template Location to mitochondria.
7. gene editing method as claimed in claim 6, it is characterized in that adopting Lipofectamine3000 transfection reagent that the DNA nuclease of mitochondria location, the sgRNA of mitochondria location and the expression vector cotransfection of RNA recovery template are entered to 293 clones, 37 degree CO2gas incubators are cultivated 48 hours. Extract cell genomic dna, adopt the specific forward primer of HA and the people source specific reverse primer of ND4 to carry out PCR.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755059A (en) * | 2016-11-08 | 2017-05-31 | 安徽省农业科学院水稻研究所 | It is a kind of for the key plasmid vector of genetic engineering and application |
| WO2017222834A1 (en) * | 2016-06-10 | 2017-12-28 | City Of Hope | Compositions and methods for mitochondrial genome editing |
| WO2018033110A1 (en) * | 2016-08-19 | 2018-02-22 | 苏州兰希亚生物科技有限公司 | Method for repairing gene point mutation |
| CN108410906A (en) * | 2018-03-05 | 2018-08-17 | 淮海工学院 | A kind of CRISPR/Cpf1 gene editing methods being applicable in Yu Haiyang shell-fish mitochondrial genomes |
| CN108513582A (en) * | 2015-06-18 | 2018-09-07 | 布罗德研究所有限公司 | Novel C RISPR enzymes and system |
| CN108595914A (en) * | 2018-05-16 | 2018-09-28 | 湖南农业大学 | One grows tobacco mitochondrial RNA (mt RNA) editing sites high-precision forecasting method |
| CN109415725A (en) * | 2016-07-01 | 2019-03-01 | 微软技术许可有限责任公司 | Molecular state machine |
| CN109853046A (en) * | 2018-11-13 | 2019-06-07 | 云南中烟工业有限责任公司 | A kind of method in rapid build CRISPR/Cas9 gene editing carrier library |
| CN111263810A (en) * | 2017-08-22 | 2020-06-09 | 纳匹基因公司 | Organelle genome modification using polynucleotide directed endonucleases |
| CN111560392A (en) * | 2020-05-07 | 2020-08-21 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | MiRNA expression vector and application thereof |
| CN112251468A (en) * | 2020-10-22 | 2021-01-22 | 钟刚 | Mitochondrial targeted gene editing complex, preparation method and application thereof, and mitochondrial genome editing method |
| US11053482B2 (en) | 2015-04-16 | 2021-07-06 | Wageningen Universiteit | Vector systems for nuclease-mediated genome editing |
| US11286478B2 (en) | 2016-04-19 | 2022-03-29 | The Broad Institute, Inc. | Cpf1 complexes with reduced indel activity |
| US11359234B2 (en) | 2016-07-01 | 2022-06-14 | Microsoft Technology Licensing, Llc | Barcoding sequences for identification of gene expression |
| US11634755B2 (en) | 2015-06-18 | 2023-04-25 | The Broad Institute, Inc. | Crispr enzymes and systems |
-
2016
- 2016-01-19 CN CN201610035146.9A patent/CN105602993A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| AREUM JO ET AL: "Efficient Mitochondrial Genome Editing by CRISPR/Cas9", 《BIOMED RESEARCH INTERNATIONAL》 * |
| HONG YU ET AL: "Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model", 《PNAS》 * |
| HUI YANG ET AL: "One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering", 《CELL》 * |
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| US11053482B2 (en) | 2015-04-16 | 2021-07-06 | Wageningen Universiteit | Vector systems for nuclease-mediated genome editing |
| US11760985B2 (en) | 2015-04-16 | 2023-09-19 | Wageningen Universiteit | CRISPR-Cas complex |
| US11479761B2 (en) | 2015-04-16 | 2022-10-25 | Wageningen Universiteit | Nuclease-mediated genome editing |
| US11781172B2 (en) | 2015-06-18 | 2023-10-10 | The Broad Institute, Inc. | Crispr enzymes and systems |
| CN108513582A (en) * | 2015-06-18 | 2018-09-07 | 布罗德研究所有限公司 | Novel C RISPR enzymes and system |
| US11773432B2 (en) | 2015-06-18 | 2023-10-03 | The Broad Institute Inc. | CRISPR enzymes and systems |
| US11634755B2 (en) | 2015-06-18 | 2023-04-25 | The Broad Institute, Inc. | Crispr enzymes and systems |
| CN108513582B (en) * | 2015-06-18 | 2022-08-05 | 布罗德研究所有限公司 | Novel CRISPR enzymes and systems |
| US11286478B2 (en) | 2016-04-19 | 2022-03-29 | The Broad Institute, Inc. | Cpf1 complexes with reduced indel activity |
| WO2017222834A1 (en) * | 2016-06-10 | 2017-12-28 | City Of Hope | Compositions and methods for mitochondrial genome editing |
| US11779657B2 (en) | 2016-06-10 | 2023-10-10 | City Of Hope | Compositions and methods for mitochondrial genome editing |
| US11422832B2 (en) | 2016-07-01 | 2022-08-23 | Microsoft Technology Licensing, Llc | Molecular state machines using HDR template insertion |
| CN109415725A (en) * | 2016-07-01 | 2019-03-01 | 微软技术许可有限责任公司 | Molecular state machine |
| US11359234B2 (en) | 2016-07-01 | 2022-06-14 | Microsoft Technology Licensing, Llc | Barcoding sequences for identification of gene expression |
| WO2018033110A1 (en) * | 2016-08-19 | 2018-02-22 | 苏州兰希亚生物科技有限公司 | Method for repairing gene point mutation |
| CN106755059A (en) * | 2016-11-08 | 2017-05-31 | 安徽省农业科学院水稻研究所 | It is a kind of for the key plasmid vector of genetic engineering and application |
| CN106755059B (en) * | 2016-11-08 | 2020-02-18 | 安徽省农业科学院水稻研究所 | Backbone plasmid vector for genetic engineering and application |
| US11920140B2 (en) | 2017-08-22 | 2024-03-05 | Napigen, Inc. | Organelle genome modification using polynucleotide guided endonuclease |
| CN111263810A (en) * | 2017-08-22 | 2020-06-09 | 纳匹基因公司 | Organelle genome modification using polynucleotide directed endonucleases |
| CN108410906A (en) * | 2018-03-05 | 2018-08-17 | 淮海工学院 | A kind of CRISPR/Cpf1 gene editing methods being applicable in Yu Haiyang shell-fish mitochondrial genomes |
| CN108595914A (en) * | 2018-05-16 | 2018-09-28 | 湖南农业大学 | One grows tobacco mitochondrial RNA (mt RNA) editing sites high-precision forecasting method |
| CN108595914B (en) * | 2018-05-16 | 2021-06-25 | 湖南农业大学 | High-precision prediction method for tobacco mitochondrial RNA editing sites |
| CN109853046A (en) * | 2018-11-13 | 2019-06-07 | 云南中烟工业有限责任公司 | A kind of method in rapid build CRISPR/Cas9 gene editing carrier library |
| CN111560392A (en) * | 2020-05-07 | 2020-08-21 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | MiRNA expression vector and application thereof |
| CN111560392B (en) * | 2020-05-07 | 2022-03-01 | 广州市妇女儿童医疗中心（广州市妇幼保健院、广州市儿童医院、广州市妇婴医院、广州市妇幼保健计划生育服务中心） | MiRNA expression vector and application thereof |
| CN112251468B (en) * | 2020-10-22 | 2023-04-04 | 钟刚 | Mitochondrial targeted gene editing complex, preparation method and application thereof, and mitochondrial genome editing method |
| CN112251468A (en) * | 2020-10-22 | 2021-01-22 | 钟刚 | Mitochondrial targeted gene editing complex, preparation method and application thereof, and mitochondrial genome editing method |
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