CN107362372B - Use application of the CRISPR technology in coronary atherosclerotic heart disease - Google Patents
Use application of the CRISPR technology in coronary atherosclerotic heart disease Download PDFInfo
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Abstract
The present invention provides a kind of method that microRNA-8086 gene is specifically knocked out using CRISPR-Cas9, the method are as follows: (1) constructs Cas9 expression vector；(2 building sgRNA expression vectors;(3) Cas9, sgRNA- are mixed into mixed liquor, inject mammal fertilized eggs, realized the purpose of the gene site-directed knockout of microRNA-8086.
Description
Technical field
The present invention relates to application of the CRISPR technology in coronary atherosclerotic heart disease, belong to medicine bioengineering skill
Art field.
Background technique
Coronary atherosclerotic heart disease is that coronary artery occurs atherosclerotic lesion and causes blood vessel
Chamber stenosis or occlusion causes heart disease, commonly referred to as " coronary heart disease " caused by myocardial ischemia, anoxic or necrosis.But it is preced with
The range of heart trouble may further include more extensively that inflammation, embolism etc. lead to luminal stenosis or occlusion.The World Health Organization is by coronary heart disease
It is divided into 5 major class: silent ischemia (latent coronary heart disease), angina pectoris, myocardial infarction, ischemic heart failure (ischemic
Heart disease) and 5 kinds of Clinical types of sudden death.Usually it is divided into stable coronary heart disease and acute coronary syndrome in clinic.
Typical chest pain is mainly reflected in clinical manifestation.Because physical exertion, excited etc. induce, sense pareordia pain of dashing forward
Bitterly, mostly ictal colic pain or squeezing pain, can also be sense of feeling oppressed.Pain is radiated to a left side since after breastbone or pareordia upwards
Shoulder, arm or even little finger of toe and the third finger, rest or buccal nitroglycerin can be relieved.Pectoralgia release position can also refer to neck, under
Jaw, tooth, abdomen etc..Pectoralgia may also appear under rest state or night, and caused by coronary spasm, also referred to as the anomaly heart is twisted
Bitterly.If pectoralgia property changes, such as the progressive pectoralgia that occurs recently, the threshold of pain gradually decline so that pause physical exertion or
Excited or even rest or while sleeping soundly, can also break out.Pain gradually aggravates, frequency conversion, and duration extension is dispelled inducement or contained
Taking nitroglycerin cannot alleviate, and often suspect unstable angina at this time.
Currently, treatment coronary atherosclerotic heart disease mainly uses antianginal drug, such as nitrate esters, kidney
Upper parathyrine beta receptor blocking agent, calcium channel blocker etc..It is disclosed in CN105816722A and a kind of treats coronary atherosclerosis
The cardiopathic Chinese materia medica preparation of property, bulk pharmaceutical chemicals include Rhizoma Chuanxiong, peach kernel, safflower, radix paeoniae rubra, Radix Angelicae Sinensis, ground bettle, aspongopus, radix cyathulae,
Red yeast rice, raw hawthorn, male semen sojae atricolor, pangolin, rhizoma corydalis, prepared RHIZOMA CYPERI with vinegar, ginkgo leaf, dalbergia wood, fushen, Radix Polygalae, smoked jujube, rhizoma nardostachyos, amber,
Cairo morningglory root or leaf, radix bupleuri, pueraria lobata, campanulaceae, cuttlefish fish ink sac.The Chinese materia medica preparation is directed to etiology and pathogenesis prescription medicine, has diagnosis and treatment
Advantage, overcome traditional Western medicine card type cannot be distinguished and carry out verification treatment, can be to a certain extent by for oral administration of Chinese medicine
The use of Western medicine is substituted, drug dependence and side effect etc. are reduced.Treatment coronary heart disease and the heart are disclosed in 101332246 B of CN
The Chinese materia medica preparation of colic pain mainly includes Radix Salviae Miltiorrhizae 18-22, Radix Notoginseng 4-6, radix paeoniae rubra 17-23, dalbergia wood 13-18, corydalis tuber 13-18, safflower 7-
11, fleece-flower root 18-22, Caulis Spatholobi 25-35, myrrh 18-22, ginseng 13-17, cortex cinnamomi 13-17, Herba Epimedii 18-22, pilose antler 4-6,
Ganoderma lucidum 11-15, cordyceps sinensis 7-9, Rhizoma Chuanxiong 12-18, peach kernel 8-12, olibanum 13-17, scorpio 4-6, centipede 4-6, root bark of tree peony 12-17
Etc. components, it is certain according to having the effect of from the point of view of experimental result.
In the prior art, it discloses in 103877576 B of CN with Card3 knock out mice and heartspecific
Card3 transgenic mice is object, by blocking mouse heart ramus descendens anterior arteriae coronariae sinistrae that myocardial infarction model is caused to be ground
Study carefully, the results showed that compared with WT mouse, Card3 knock out mice cardiac infarction ratio, the degree of myocardial hypertrophy and fibrosis
Obvious to be suppressed, heart function is clearly better；With GDF1 knock out mice and heartspecific GDF1 in 103893743 B of CN
Transgenic mice is experimental subjects, by blocking mouse heart ramus descendens anterior arteriae coronariae sinistrae that myocardial infarction model is caused to be ground
Study carefully, the results showed that compared with MEM-Cre control mice, GDF1 knock out mice cardiac infarction ratio, myocardial hypertrophy and fiber
The degree of change obviously increases, and heart function obviously deteriorates；And cardiac infarction ratio, the cardiac muscle of heartspecific GDF1 transgenic mice
Plump and fibrosis degree is obviously suppressed, and heart function is obviously improved.It discloses in 103898189 B of CN with SHPS-1 base
Because knock-out mice and heartspecific SHPS-1 transgenic mice are experimental subjects, before blocking mouse heart arteria coroaria sinistra
Descending branch (LAD) causes myocardial infarction model, the results showed that compares with WT control mice, SHPS-1 knock out mice is postoperative in MI
The degree of cardiac infarction ratio, myocardial hypertrophy and fibrosis is obviously suppressed, and heart function is clearly better；In 103893763 B of CN
It discloses using Vinexin- β knock out mice and heartspecific Vinexin- β transgenic mice as experimental subjects, passes through resistance
Disconnected mouse heart ramus descendens anterior arteriae coronariae sinistrae (LAD) causes myocardial infarction model, the results showed that it is compared with WT control mice,
Vinexin- β knock out mice is obviously suppressed in the degree of MI postoperative cardiac infarct ratio, myocardial hypertrophy and fibrosis, the heart
Function is clearly better.By result above it can be found that coronary atherosclerotic heart may be implemented by gene interference
The treatment of disease.
Currently, rule repeats system (clustered regularly interspaced short at the short palindrome in race interval
palindromic repeat；CRISPR-associated, CRISPR_Cas9) it is a kind of answering with endonuclease activity
Zoarium, identifies specific DNA sequence dna, carry out specific site cutting cause double-strand DNA cleavage (Double-strand breaks,
DSB), under conditions of no template, the non-homogeneous recombination end connection of generation (Non-homologous end joining,
NHEJ), frameshift mutation (frameshift mutation) is caused, gene knockout is caused.This technology due to can quickly, it is easy,
Efficiently any gene of target gene group started to bud out into popularity as explosion in 2012 so as to cause extensive concern.
Since its is easy to operate, can target multiple genes simultaneously, can advantages, the Cas9 such as high-throughput preparation, low cost have become
A kind of technology with fastest developing speed.Just because of its superiority, this technology is ranked in the 20130 big progress that Nature recommends
First.
It is by two kinds of tiny RNAs that Cas9, which targets cutting DNA, -- crRNA (CRISPR RNA) and tracrRNA (trans-
Activating crRNA) and target sequence complementation identification principle realize.Two kinds of tiny RNAs one has been fused into now
RNA chain, abbreviation sgRNA (single guide RNA).Therefore, can sgRNA accomplish that specificity, accurate targeting target gene are
CRISPR-Cas9 can specific knockdown target gene prerequisite, either miss the target or mistake targeting, can all influence
Specific knockdown of the CRISPR-Cas9 to target gene.Therefore, it can design, prepare accuracy and selectively targeted target
The sgRNA of gene becomes the key technology of CRISPR-Cas9 gene knockout.Compared with ZFN and siRNA, CRISPR-Cas9 has
More rapidly, easy, efficient, multidigit point, the selectively targeted advantage for knocking out gene.
Summary of the invention
According in a first aspect, providing hprt minigene acid miR- the purpose of the present invention is aiming at the shortcomings in the prior art
8086 purposes knocked out using CRISPR/Cas9 system.This be for the first time using CRISPR/Cas9 systemic characteristic general
The miR-8086 of micrRNA family is knocked out.The purpose that miR is knocked out can be efficiently realized using this new method.
Still further aspect of the present invention obtains miR-8086's and Card3 and SHPS-1 by screening according to applicant's early period
Positive Expression modulation relationship provides the expression of inhibition Card3 and SHPS-1 gene after the knockout of miR-8086, to treat hat
Shape atherosclerotic heart disease.
The present invention additionally provides a kind of sgRNA, sequence is according to miR-8086 precursor sequence, and by early period, more than 40 are set
There is the sgRNA for preferably knocking out effect by the only one that preliminary test is found, sequence is such as in the sgRNA target spot of meter
Gcacagccttggtgtctctagtcc (shown in SEQ ID NO:1).
The present invention provides a kind of method that microRNA gene is specifically knocked out using CRISPR-Cas9, the method are as follows:
(1) Cas9 expression vector is constructed；(2 building sgRNA expression vectors；(3) Cas9, sgRNA- are mixed into mixed liquor, inject lactation
Class fertilised non-human eggs are realized the purpose of the gene site-directed knockout of microRNA.
Compared with prior art, the beneficial effects are mainly reflected as follows:
1) gene modification efficiency is higher, reduces the unreliability of traditional technology；2) operating technology is simple, without by multiple
The series of steps such as miscellaneous targeting vector building, ES cell screening, allophenic mice breeding；It 3) can be real by a simple step
Existing complicated cardiopathic treatment, has great market application prospect.
Detailed description of the invention
Fig. 1 lentiCRISPR v2 carrier figure.
Fig. 2 (1), (2), (3) are respectively the mrna expression amount pair before and after miR-8086, Card3 and SHPS-1 gene knockout
Than figure.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Material therefor, reagent etc. commercially obtain unless otherwise instructed in following embodiments.
1 design of primers of embodiment
1, the outer Cas9 expression vector of construct, is named as pGEM-Cas9, is responsible for synthesizing by winning profit biology, sequence is this
The common sequence in field.
2, vector construction and the preparation of sgRNA skeetal coding sequence is transcribed in vitro.
SgRNA is designed for mmu-miR-8086 gene, and positive oligonucleotide sequence and reverse oligonucleotide sequence can be with
It is complementarily shaped to the double chain DNA fragment with cohesive end:
F:CACCGgcacagccttggtgtctctagtcc
R:CggactagagacaccaaggctgtgcCAAA。
Embodiment 2, the sgRNA expression vector for constructing mmu-miR-8086 gene
1. synthetic DNA Insert Fragment
(1) the forward and reverse oligonucleotide sequence of above-mentioned design is synthesized
Oligonucleotide sequence can specifically be synthesized by commercialized company (Shanghai Sangon Biotech Company) according to the sequence of offer.
By the annealing of corresponding forward and reverse oligonucleotide sequence, renaturation, the double-stranded DNA piece with cohesive end is formed
Section.
Reaction system (20 μ L) is as follows:
Positive oligonucleotides (10 μM): 1 μ L
Reverse oligonucleotide (10 μM): 1 μ L
10 × PCR buffer:2 μ L
ddH2O:16 μ L
Above-mentioned reaction system is put into PCR instrument, and is reacted by following procedure.
Response procedures:
95℃,5min；
80℃,5min；
70℃,5min；
59℃,5min；
50℃,5min；
Naturally it is down to room temperature.
2. constructing sgRNA expression vector
(1) BsmB I digestion with restriction enzyme destination carrier lentiCRISPR v2 plasmid (excellent precious biology, goods are utilized
Number: VT8107).
It is prepared according to following reaction system:
LentiCRISPR v2 plasmid: 1 μ g
10 × digestion buffer:2 μ L
BsmB I restriction enzyme: 2 μ L
Supplement ddH2O to 20 μ L of total volume
Endonuclease reaction system is placed in 37 DEG C of reaction 3h.
(2) electrophoretic separation and cmy vector segment
After digestion, digestion mixture is separated by agarose gel electrophoresis, selects carrier segments (about
It 12kb) is cut, and is recycled by DNA gel recovery column.
(3) double chain DNA fragment of synthesis and carrier main leaf section are attached and convert Escherichia coli
The double chain DNA fragment that renaturation obtains is attached with the carrier segments that recycling obtains and is reacted, according to following reaction
System is prepared:
LentiCRISPR v2 carrier segments: 100ng
Double chain DNA fragment: 200ng
T4 ligase: 1 μ L
Buffer:1 μ L is reacted in T4 connection
Supplement ddH2O to 10 μ L of total volume
Connection mixture is placed in 25 DEG C of reaction 2h.
Connection mixture is converted e.colistraindh5α after reaction: it is big that 100 μ L are added into connection mixture
Enterobacteria DH5 α competent cell, is incubated for 30min on ice；Mixture is put into 42 DEG C of water-baths, is put into after heat shock 90s cold on ice
But；100 μ L LB culture mediums, 37 DEG C of shaking table culture 20min are added to mixture；Mixture is applied into Amp LB plate, 37 DEG C of cultures
14h。
(4) correct transformed clone is identified
Several bacterium colonies are selected from Amp LB plate to expand culture, and are extracted plasmid and are carried out digestion identification.Select possibility
Correctly clone is sequenced, and by sequencing, discovery insetion sequence is correct.Correct lentiCRISPR v2-sgRNA is carried
Body clone carries out conservation, and extracts corresponding plasmid.
Embodiment 3, transgenic mice preparation
1) it is as follows to inject mouse system by CRISPR/Cas9:
| pGEM-Cas9 | 50ng/μl |
| lentiCRISPR v2-sgRNA | 10ng/μl |
| Mixed population product | 20μl |
2) it injects
Using Eppendorf2xTransferManNK2 microinjection instrument draw 2 μ l step 1) mixed liquors inject 60 by
Smart ovum.Then become pregnant.
After mouse is born 5 days, clip mouse nail extracts genomic DNA.PCR identify miR-8086 and Card3 and
SHPS-1 gene expression dose is only used as compareing not carry out the mouse 30 of gene knockout.As shown in Fig. 2, compared with the control, it is small
Relative to control, expression is reduced to for the intracorporal miR-8086 of mouse and Card3 and SHPS-1 gene expression dose
0.2%, 11.7%, 12.5%.This absolutely proves that the intracorporal miR-8086 of mouse is completely knocked out, and gene expression is
Almost lost.Corresponding, Card3 and SHPS-1 gene expression dose reduces also significantly.
60 mouse are dissected, miR-8086 knock out after mouse, heart significantly becomes smaller, average external volume be do not knock out it is small
The 65.2% of mouse.This is absolutely proved, same to can be used for doing physical therapy the coronary atherosclerotic heart by the knockout of miR-8086
Popular name for.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention
Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence table
<110>Hou Dongxue
<120>application of the CRISPR technology in coronary atherosclerotic heart disease is used
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
gcacagcctt ggtgtctcta gtcc 24
<210> 2
<211> 29
<212> DNA
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
caccggcaca gccttggtgt ctctagtcc 29
<210> 3
<211> 29
<212> DNA
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
cggactagag acaccaaggc tgtgccaaa 29
Claims (3)
1.miR-8086's knocks out application of the reagent in the reagent that Card3 and SHPS-1 gene expression is lowered in preparation；Wherein institute
Coronary atherosclerotic heart disease can be treated by stating reagent.
The knockout reagent of 2.miR-8086 is preparing the purposes for treating the drug of coronary atherosclerotic heart disease.
3. purposes as claimed in claim 2 uses CRISPR/CAS technology wherein knocking out, wherein the sgRNA sequence such as SEQ used
Shown in ID NO:1.
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| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
| US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9340799B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | MRNA-sensing switchable gRNAs |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| EP3177718B1 (en) | 2014-07-30 | 2022-03-16 | President and Fellows of Harvard College | Cas9 proteins including ligand-dependent inteins |
| EP3365356B1 (en) | 2015-10-23 | 2023-06-28 | President and Fellows of Harvard College | Nucleobase editors and uses thereof |
| GB2568182A (en) | 2016-08-03 | 2019-05-08 | Harvard College | Adenosine nucleobase editors and uses thereof |
| AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| KR102622411B1 (en) | 2016-10-14 | 2024-01-10 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | AAV delivery of nucleobase editor |
| WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| WO2018165629A1 (en) | 2017-03-10 | 2018-09-13 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| EP3601562A1 (en) | 2017-03-23 | 2020-02-05 | President and Fellows of Harvard College | Nucleobase editors comprising nucleic acid programmable dna binding proteins |
| WO2018209320A1 (en) | 2017-05-12 | 2018-11-15 | President And Fellows Of Harvard College | Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| EP3676376A2 (en) | 2017-08-30 | 2020-07-08 | President and Fellows of Harvard College | High efficiency base editors comprising gam |
| KR20200121782A (en) | 2017-10-16 | 2020-10-26 | 더 브로드 인스티튜트, 인코퍼레이티드 | Uses of adenosine base editor |
| BR112021018606A2 (en) | 2019-03-19 | 2021-11-23 | Harvard College | Methods and compositions for editing nucleotide sequences |
| DE112021002672T5 (en) | 2020-05-08 | 2023-04-13 | President And Fellows Of Harvard College | METHODS AND COMPOSITIONS FOR EDIT BOTH STRANDS SIMULTANEOUSLY OF A DOUBLE STRANDED NUCLEOTIDE TARGET SEQUENCE |
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| WO2014100252A1 (en) * | 2012-12-18 | 2014-06-26 | University Of Washington Through Its Center For Commercialization | Methods and compositions to modulate rna processing |
| CN103877576B (en) * | 2014-04-10 | 2017-01-11 | 武汉大学 | Function and application of Caspase activation and recruitment domain 3 (Card3) gene in coronary atherosclerotic heart disease |
| CN103898189B (en) * | 2014-04-10 | 2015-10-28 | 武汉大学 | The application of signal adjusting protein alpha (SHSP-1) gene in myocardial infarction |
| CN104894068A (en) * | 2015-05-04 | 2015-09-09 | 南京凯地生物科技有限公司 | Method for preparing CAR-T cell by CRISPR/Cas9 |
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