CN106906242A - A kind of method that raising CRIPSR/Cas9 targeting knock outs gene produces nonhomologous end joint efficiency - Google Patents
A kind of method that raising CRIPSR/Cas9 targeting knock outs gene produces nonhomologous end joint efficiency Download PDFInfo
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Abstract
The invention belongs to molecular biology and field of biomedicine technology, the present invention relates to a kind of method of raising CRIPSR/Cas9 targeting knock out gene NHEJ efficiency.Specifically, present invention design filters out the small molecules interference RNA of efficiently interference pten gene expressions, and during using CRISPR/Cas9 system targeting knock out genes, the cotransfection microRNA can effectively improve the NHEJ efficiency of target gene.In cell multiple target validation, it is significantly improved, the method low cost, simple to operate, efficiency high, efficiency and application for CRISPR/Cas9 technologies have good facilitation.
Description
Technical field
The invention belongs to molecular biology and field of biomedicine technology, and in particular to one kind improves CRIPSR/Cas9 targets
Method to gene nonhomologous end engagement (Non-homologous end joining, abbreviation NHEJ) efficiency is knocked out.
Background technology
CRISPR/Cas systems are the adaptive immune system development from bacterium and Archimycetes confrontation adventitious viruses or plasmid
, including three kinds of different types, the DNA restriction endonucleases Cas9 of the wherein CRISPR/Cas systems of the types of Type II only has
One subunit, structure is the simplest, so using also most extensive.In addition to Cas9 albumen, the system also include two it is short
CRISPR RNAs（crRNAs）With trans-activating crRNAs（tracrRNA）.Ripe crRNA-tracrRNA
Complex can instruct Cas9 albumen to target sequence by base pair complementarity, and in PAM（protospacer
adjacent motif）Neighbouring specific cleavage DNA double-strands, form DSB（double strand break）.DSB can be with
It is repaired by two kinds of approach, one kind is nonhomologous end engagement（Non-Homologous End Joining NHEJ）DNA
Repair mode, another kind is homologous recombination repair（Homology Directed Repair HDR) mode.NHEJ reparation sides
Insertion or missing of the formula there may be base, so as to produce frameshift mutation, or are likely to be mutated into terminator codon, and these are dashed forward
Deformation type can change the ORFs of genes of interest；HDR modes need one section of template piece homologous with fragment is sheared
Section repairs DSB, and this repair mode can will be used as the sequence replicating of the homologous fragment of template in genes of interest,
So specific genetic fragment can be incorporated into genes of interest using this repair mode.
Cas9 sometimes poor cutting efficiency may be being associated of how repairing with DNA, because DNA repair machine
System --- any in basic house keeping enzyme DNA plerosis may cause the fracture or missing that mortality is mutated --- different cells it
Between have differences.He infers that the oligonucleotides with human genome in the absence of homology may interfere with this repair process, so as to carry
High gene knocks out success rate.
CRISPR-Cas9 gene editings may be considered the competition between cutting and DNA reparations：Once Cas9 is cut
Cut, cell accurately replaces the DNA by cutting, and subsequent Cas9 cuts this DNA by replacing again, so as to be absorbed in one kind
Endless cutting and the circulation of reparation, until these repair enzymes make a mistake, these genes finally all lose function.These few cores
Thuja acid may reduce the fidelity of this repair process, allow cell to be switched to a kind of reparation for being more prone to and occurring in other words
Journey, so as to allow Cas9 to be easier to allow gene to be broken.The present invention can greatly improve CRISPR/Cas9 and knock out effect
Rate, while making the technology more simple, easy to operate.
The content of the invention
The invention belongs to molecular biology and field of biomedicine technology, CRIPSR/Cas9 is improved the present invention relates to one kind
The method of targeting knock out gene NHEJ efficiency.Specifically, present invention design filters out the small of efficiently interference pten gene expressions
Molecule interference RNA, during using CRISPR/Cas9 system targeting knock out genes, the cotransfection microRNA can be carried effectively
The NHEJ efficiency of target gene high.In cell multiple target validation, it is significantly improved, the method low cost, simple to operate, efficiency
It is high.Efficiency and application for CRISPR/Cas9 technologies have good facilitation.
Technical solution of the present invention is as follows：
1st, the small molecules interference RNA of people's pten gene expressions, design, synthesis and screening are efficiently disturbed；
2nd, design, build the CRIPSR/Cas9 carriers for knocking out people's pten genes, and the small molecule interference that cotransfection screening is obtained
RNA, by detecting the NHEJ efficiency that target site is produced, confirms that whether CRISPR/Cas9 carrier cotransfection small molecules interference RNAs may be used
To improve NHEJ efficiency；
3rd, small molecules interference RNA is applied to multiple CRISPR/Cas9 target sites editor checkings, confirms that small molecules interference RNA is carried
The accuracy of NHEJ efficiency high.
Brief description of the drawings
In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：
Fig. 1 is the expression quantity that reverse transcription PCR detects pten genes（Wherein M is DL2000, and 1 is pten-RNAi-1, and 2 is pten-
RNAi-1,3 is pten-RNAi-3, and 4 is pten-RNAi-4, and 5 is pten-RNAi-5, PCR amplification 264bp size fragments）；
Fig. 2 is two target area PCR primer T7 Endonuclease I digestion products of Lin28a, agarose gel electrophoresis inspection
Survey result（Wherein A groups are that siRNA+PX458-Lin28A-T1, B group are siRNA+PX458-Lin28A-T5, C
Group is PX458-Lin28A-T1, and D groups are PX458-Lin28A-T5, and E groups are siRNA, and F groups are PBS）.
Specific embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.It is unreceipted specific in embodiment
The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide（The third edition, J. Pehanorm Brookers etc.
Write）Described in condition, or according to the condition proposed by manufacturer.
The CDS regions of PTEN Gene are found according to ncbi database, and combines NCBI Primer-BLAST design primer sequences
Row, while according to patent（201611252179.5）Synthesis lin28a gene target sites region amplimer, sequence is as follows：
5 sequences of small interfering RNAs for PTEN genes are designed by online software, and is synthesized, its corresponding RNA sequence is such as
SEQ ID NO .1 -- shown in SEQ ID NO .5.
Experiment specific steps
Transfect first 3 days, recovery human embryonic kidney cell（293T cell lines, Chinese Academy of Sciences's Shanghai cell bank）, cell is put into added with 10%
FBS+DMEM blake bottles in, in 37 DEG C, 5% CO2Incubator in cultivate, transfect first 1 day, Secondary Culture recovery cell；
The culture medium exhaustion in T75 bottles of 293T cells will be cultivated, 0.25% pancreatin for adding 2 mL, 4 DEG C of refrigerators to take out makes its equal
Even covering bottom of bottle, is placed in 3 ~ 5 min in 37 DEG C of incubators, takes out, and rocking can find that cell departs from bottom, and it is all shaken
Under, the 10%FBS+DMEM culture mediums preheated in 3 mL, 37 DEG C of water-baths are added, blown and beaten with 10 mL pipettes, cell is hanged
Supernatant liquid is collected, centrifugation；
Transfection is first 1 day, inoculation 2 × 104In individual 293T cells to 48 well culture plates, incubated overnight makes cell density during transfection
Reach 80%；
Experiment packet is pten-RNAi-1 transfection groups, pten-RNAi-2 transfection groups, pten-RNAi-3 transfection groups, pten-
RNAi-4 transfection groups, pten-RNAi-5 transfection groups, PBS transfection groups（Control group）Every group of 6 multiple holes；
SiRNA and transfection reagent：SiRNA 20pmol is added per hole：The μ L of transfection reagent 1.0：With 50 μ L Opti-
MEM dilutes, and gently the mixing of pressure-vaccum 3 ~ 5 times, stands 20 min at room temperature, and being subsequently added in culture dish carries out cell transfecting（In detail
Step is referring to the operation instructions of Lipofectamine 2000）, after the completion of transfection procedure, after 37 DEG C are cultivated 4h, with 10%
The complete medium of FBS+DMEM culture mediums changes liquid, continues to cultivate；
1., by cell culture to 24 h, using cell of PBS, plus Beyozol Reagent cell lysis, room temperature is put
Put 5 min；
2. often pipe adds 0.2mL chloroforms, acutely the concussion s of centrifuge tube 15, and room temperature places 3 min, and 12000g, 4 DEG C 15 is centrifuged
min。
3. supernatant is taken after being centrifuged, adds isometric isopropanol, the min of ice bath 10 that 12000g, 4 DEG C of 10 min is centrifuged；
4. remove supernatant after being centrifuged, washed with the ethanol of 1mL75%, be then centrifuged for 7400g, 4 DEG C 5 minutes；
5. ethanol solution is removed, is dried naturally 5 minutes, add DEPC water dissolving RNAs；
6. RNA mass is detected；
7. the Total RNA that will be carried as template, with Takara PrimeScript II 1st strand cDNA
Synthesis Kit article No.s 6210A/B synthesizes cDNA；
Reverse transcription operating procedure：
（1）Prepare mixed system as follows：
（2）65 DEG C of incubation 5min；Cooled on ice is placed at once；
（3）Prepare reaction system as follows：
（4）Gently mix；
（5）60 min are incubated at 42 DEG C；
（6）It is reverse transcriptase inactivation and terminating reaction that 95 DEG C are incubated 5 min.
8. 1 pair of primer is designed according to genes of interest Strn and enter performing PCR, reaction system is as follows：
9. electrophoresis, takes 5 μ L PCR primers, 1.0% agrose regular, 100V 1X TAE buffer, 20 min；
10. the expression quantity of pten genes in 5 cells of siRNA transfection treatment is judged according to electrophoresis result, such as the institute of accompanying drawing 1
Show,
Be can be seen that from PCR band brightness：2nd, 4, No. 5 bands are weaker, with obvious jamming effectiveness.
According to patent（201611252179.5）In knockout Lin28a gene Cs two target position of RISPR/Cas9 for screening
Point sequence, constructs corresponding CRIPSR/Cas9 carriers, according to patent respectively（201611252179.5）In entitled PX458-
Lin28A-T1 and PX458-Llin28A-T5, carrier construction detailed step is with reference to patent（201611252179.5）.
Cotransfection, transfects first 1 day, inoculation 2 × 104In individual 293T cells to 48 well culture plates, incubated overnight, when making transfection
Cell density reach 80%.Pten genes siRNA respectively with CRISPR/Cas9 carriers PX458-lin28A-T1 and
PX458-lin28A-T5 cotransfection people's 293T cell lines, specific experiment packet：A groups are siRNA+PX458-Lin28A-
T1, B group are that siRNA+PX458-Lin28A-T5, C group are PX458-Lin28A-T1, and D groups are PX458-Lin28A-
T5, E group are siRNA, and F groups are PBS.
Configuration RNA and transfection reagent：The pmol of siRNA 20 is added per hole：Transfection reagent=1.0 μ L：With 50 μ L
Opti-MEM dilutes, and gently 3 ~ 5 mixings of pressure-vaccum, stand 20 min at room temperature.
PX458-Lin28A-T1/ PX458-Lin28A-T5：Transfection reagent=0.8 μ g：2 μ L are dilute with 50 μ LOpti-MEM
Release, gently pressure-vaccum is mixed, and 20 min are stood at room temperature.
According to experiment packet, using above-mentioned compound method by siRNA and plasmid transfection 293T cells.
After the completion of transfection procedure, after 37 DEG C are cultivated 4 h, liquid is changed with the complete medium of 10%FBS+DMEM culture mediums,
Continue to cultivate.Treat to come into force cell culture to 24 h rate.
It is template with the DNA for extracting, expands target sequence, identified using T7 Endonuclease I digestions, verifies target spot
Series jump situation.
PCR reaction systems are as follows：
PCR amplification programs：95 DEG C of min of predegeneration 3；95 DEG C of denaturation 30 s, 58 DEG C of annealing 30 s, 72 DEG C of 40 s of extension, 30 are followed
72 DEG C of 5 min of extension, last 4 DEG C of insulations after ring.
PCR primer 37 DEG C of water-bath digestion 1h of T7 Endonuclease I, digestion system is as follows：
Digestion products are tested and analyzed through agarose gel electrophoresis, fruit Fig. 2 display each groups（A groups, B groups, C groups, D groups, E groups and F groups）
Target site NHEJ efficiency, the gray value InDel (%) of software analysis band：A groups 7.8%, B groups 8.6%, C groups are that 2.3%, D groups are
1.1%, siRNA is distinguished for the formation NHEJ efficiency of two target sites of CRISPR/Cas9 targeting knock out Lin28a genes
Improve 3.39 times and 7.8 times.
SEQUENCE LISTING
<110>Chongqing Gao Sheng biological medicines Co., Ltd
<120>A kind of method that raising CRIPSR/Cas9 targeting knock outs gene produces nonhomologous end joint efficiency
<130> 2017
<160> 10
<170> PatentIn version 3.3
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<211> 19
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<213>Artificial sequence
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Claims (6)
1., the present invention relates to a kind of CRISPR/Cas9 technologies that can effectively improve in the method for knocking out gene application, its feature exists
In：While transfection CRISPR/Cas9 carriers, the small molecules interference RNA being related in the cotransfection present invention can be effectively improved
The efficiency of the NHEJ that CRISPR/Cas9 systems target site is formed, specifically includes following steps：
（1）Design the gRNA of pten genes, 3 target sites of each gene synthesize corresponding oligo and with BbsI digestions
PX458 carriers are attached, and connection product is transformed into escherichia coli DH5a, are coated with amicillin resistance
On LB flat boards, the positive bacterium colony of screening extracts positive bacterium colony plasmid and is analyzed and is sequenced, and is confirming gRNA expression vector establishments just
Really；
（2）By step（1）The gRNA carriers of structure transfect 293T cells with siRNA cotransfection, and the gRNA for separately taking structure is carried
Body transfects 293T cells（As a control group）；
（3）By step（2）Transfection 24 ~ 48 hours, cell filters out the transfection positive cells of GFP marks through airflow classification；
（4）Genome is extracted, PCR amplification target site region segments anneal PCR primer, and use T7E1 digestions, using agarose
Detected through gel electrophoresis, and carry out gray analysis（indel）Detection NHEJ efficiency, testing result finds that cotransfection siRNA can
It it is 3.39 ~ 7.8 times effectively improve CRISPR/Cas9 to form NHEJ efficiency in target site.
2. small molecules interference RNA according to claim 1, it is characterised in that：The microRNA has effectively lower mediator
The expressive function of pten genes.
3. small molecules interference RNA according to claim 1, its corresponding RNA sequence such as sequence table SEQ ID NO. 3-4,
Shown in SEQ ID NO. 7-8 and SEQ ID NO. 9-10.
4. small molecules interference RNA according to claim 1, be 3 microRNA sequences described in claim 3 etc.
Compare mixture.
5. siRNA of the present invention can effectively improve CRISPR/Cas9 systems targeting knock out people in 293T cells
The NEHJ efficiency of pten genes.
6. the technical method of the efficient targeting knock out people pten genes of a kind of CRISPR/Cas9.
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Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| CN108504657A (en) * | 2018-04-12 | 2018-09-07 | 中南民族大学 | The method for knocking out HEK293T cell KDM2A genes using CRISPR-CAS9 technologies |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| 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) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
-
2017
- 2017-03-16 CN CN201710155251.0A patent/CN106906242A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JIAWEI GUAN等: "Nuclear PTEN interferes with binding of Ku70 at double-strand breaks through post-translational poly(ADP-ribosyl)ation", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| XIN HAN等: "CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation", 《SCI. ADV》 * |
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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 |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
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