CN109957570A - The gRNA sequence and its application of targeting editor's bcr-abl fusion - Google Patents

The gRNA sequence and its application of targeting editor's bcr-abl fusion Download PDF

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CN109957570A
CN109957570A CN201910272330.9A CN201910272330A CN109957570A CN 109957570 A CN109957570 A CN 109957570A CN 201910272330 A CN201910272330 A CN 201910272330A CN 109957570 A CN109957570 A CN 109957570A
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grna
bcr
sequence
abl
seq
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黄峥兰
骆贞红
高淼
冯文莉
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Chongqing Medical University
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Abstract

The present invention provides the gRNA sequence and its application of targeting editor's bcr-abl fusion, pass through three sections of special target sequence site DNA fragmentations in positioning bcr-abl gene, and gRNA expression plasmid is constructed for this specific site, by this plasmid and FokI-dCas9 expression plasmid and donor nuclear transfection K562 cell, it can make bcr-abl gene that selective cleavage occur, and homologous reparation is carried out by template of donor dna, insert it into the destruction that 8 bases eventually lead to bcr-abl gene.Significant advantage of the invention shows that the RFNs for target sequence site building special in bcr-abl sequence can efficiently target bcr-abl gene and DNA double chain fracture occurs, repair with HDR mode makes bcr-abl that the mutation such as frameshit occur and is destroyed, to lose the vicious transformations potential such as rush proliferation, suppression apoptosis.

Description

The gRNA sequence and its application of targeting editor's bcr-abl fusion
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of gRNA of targeting editor bcr-abl fusion Sequence and its application.
Background technique
Chronic myelocytic leukemia (Chronic myeloid leukemia, CML) is initiated by the one of candidate stem cell Class malignant clone proliferative diseases, root of causing a disease are that No. 9 chromosomes and No. 22 translocations form bcr-abl fusion Gene.The fusion can encode the BCR-ABL fusion protein with strong tyrosine kinase activity, which continues Downstream JAK-STAT is activated, the signal paths such as MEK-ERK, CRKL promote undesired cell proliferation and inhibit its apoptosis.Clinically make Good effect is achieved with tyrosine kinase inhibitor (tyrosine kinase inhibitors, TKIs) treatment CML, but Being patient still more than 25% generates drug resistance to TKIs, and CML stem cell it is insensitive to TKIs be CML recurrence a weight Want reason.Therefore, it needs to explore new treatment method, keeps it equally effective to CML drug resistance or recurrence patient.
Bcr-abl fusion is the principal element that CML occurs and recurs.In theory, it is destroyed from gene Bcr-abl will block the translation of BCR-ABL, to thoroughly treat CML.Gene editing can be realized having by force for gene site-directed transformation The tool of power, is widely used in gene therapy.The regular short palindrome repetition in cluster interval/Cas9 nuclease (clustered Regularly interspaced short palindromic repeats/CRISPR-associated systems9, It CRISPR/Cas9 is) new after Zinc finger nuclease (ZFNs) and class activating transcription factor effector nuclease (TALENs) Type genome pinpoints renovation technique.CRISPR/Cas9 nuclease under the guidance of single guided RNA (sgRNA), Spacer sequence plays the fixed point dissection to target DNA in conjunction with target gene base pair complementarity, by Cas9 nuclease.Fracture The donor dna that is added using external source of DNA double chain as template, triggering with source orientation reparation (homology directed repair, HDR it), or in non-homologous end joining (nonhomologous end joining, the NHEJ) mode easily to malfunction is repaired It is multiple.Wherein HDR can carry out the operation such as fixed point insertion, missing, amendment to target gene, and NHEJ is due to easily malfunctioning, it is easy to lead Cause target gene that insertion, missing and frameshift mutation occurs, to realize the destruction (gene disruption) to target gene.With ZFNs is compared with TALENs, and CRISPR/Cas9 has low to target-gene sequence feature request, and assembly is simple, and modification rate is high, for The advantages that different loci easy switching, still, CRISPR/Cas9 relatively poor specificity, limit it in field of gene Application.
Summary of the invention
Goal of the invention:
An object of the present disclosure is to provide a kind of gRNA sequence of targeting editor bcr-abl fusion.
It is a further object of the present invention to provide the methods of above-mentioned targeting editor's bcr-abl fusion.
It is yet another object of the invention to provide the expression vectors for containing above-mentioned gRNA sequence.
It is yet another object of the invention to provide above-mentioned gRNA sequences in preparation prevention or treatment chronic myelocytic leukemia medicine Application in object.
The technical scheme is that a kind of gRNA sequence of targeting editor bcr-abl fusion, including gRNA-15, GRNA-16 or/and gRNA-17 sequence, the gRNA-15 sequence include left subluxation point sequence SEQ ID NO:1 and right half site Sequence SEQ ID NO:2；The gRNA-16 sequence includes left subluxation point sequence SEQ ID NO:3 and right subluxation point sequence SEQ ID NO:4；The gRNA-17 sequence includes left subluxation point sequence SEQ ID NO:5 and right subluxation point sequence SEQ ID NO:6.
Specifically, SEQ ID NO:1 and SEQ ID NO:2 sequence is as follows:
Sequence names SEQ ID NO:1 SEQ ID NO:2
gRNA-15 GTAGCATCTGACTTTGAGCC AGCCGCTCGTTGGAACTCCA
。
Specifically, SEQ ID NO:3 and SEQ ID NO:4 sequence is as follows:
Sequence names SEQ ID NO:3 SEQ ID NO:4
gRNA-16 TTCAGCGGCCAGTAGCATCT GTCTGAGTGAAGCCGCTCGT
。
Specifically, SEQ ID NO:5 and SEQ ID NO:6 sequence is as follows:
Sequence names SEQ ID NO:5 SEQ ID NO:6
gRNA-17 TTTCGTTGCACTGTATGATT AACACTCTAAGCATAACTAA
。
Preferably, the present invention targets the gRNA sequence of editor's bcr-abl fusion, and the sequence is gRNA-17, described GRNA-17 sequence includes left subluxation point sequence SEQ ID NO:5 and right subluxation point sequence SEQ ID NO:6 sequence.
The method that the present invention targets editor's bcr-abl fusion, the FokI nuclease (RNA guided with CRISPR RNA Guided FokI nucleases, RFNs) specificity cutting CML Disease-causing gene bcr-abl, promote bcr-abl gene to occur DNA double chain is broken, and the NotI restriction enzyme site of 8 bases is introduced in donor dna, and the bcr-abl of fracture is using donor dna as mould Plate carries out homologous reparation, causes bcr-abl gene that frameshift mutation occurs after inserting it into 8 bases, so as to cause bcr-abl base The destruction of cause.
The present invention is by gRNA by way of nuclear transfection, and the RFNs and donor of FokI-dCas9 expression vector composition are simultaneously It is transfected into K562 suspension cell, routine culture 60 hours.By extracting cell genomic dna, DNA break occurs for PCR amplification And the segment of homologous recombination, digestion identification is carried out with NotI, the NotI enzyme for whether being inserted into 8 bases in cellular genome detected Then plasmid is transferred to K562 cells play dissection by way of nuclear transfection by enzyme site, destroying bcr-abl gene can press down BCR-ABL protein expression processed, detects GAP-associated protein GAP by Western Blot and colony formation, FCM detects its effect.
Upstream primer (Forward) sequence that the PCR amplification uses is as shown in SEQ ID NO:7, downstream primer (Reverese) sequence is as shown in SEQ ID NO:8.
Specifically, SEQ ID NO:7 and SEQ ID NO:8 sequence is as follows:
The present invention contains the expression vector of the gRNA sequence of targeting editor's bcr-abl fusion, which contains GRNA sequence fragment described in gRNA-15, gRNA-16 or/and gRNA-17.
Wherein above-mentioned expression vector plasmid is PSQT1313 (Addgene#53370).The expression plasmid of FokI-dCas9 is PSQT1601(Addgene#53369)。
Application of the above-mentioned gRNA sequence in preparation prevention or treatment chronic myelocytic leukemia drug.
Beneficial effect
The present invention located three sections of special target sequence site DNA fragmentations in bcr-abl gene, and be directed to this specific site Bcr- can be made for this plasmid and FokI-dCas9 expression plasmid and donor nuclear transfection K562 cell by constructing gRNA expression plasmid Selective cleavage occurs for abl gene, and carries out homologous reparation by template of donor dna, inserts it into 8 bases and eventually leads to bcr- The destruction of abl gene.Significant advantage of the invention is shown for target sequence site building special in bcr-abl sequence RFNs can efficiently target bcr-abl gene and DNA double chain fracture occurs, and repair with HDR mode keeps bcr-abl generation frameshit prominent Become and be destroyed, to lose the vicious transformations potential such as rush proliferation, suppression apoptosis, it was demonstrated that RFNs targeting destroys bcr-abl gene and kills Wound and the feasibility for inhibiting CML cell.
Detailed description of the invention
Fig. 1 is PCR product sequencing result peak value figure；It is the NotI restriction enzyme site of insertion shown in its center；
Fig. 2 is Western Blot detection K562 cell BCR-ABL expression figure；
Fig. 3 is Western Blot detection K562 cell γ H2AX expression figure；
Fig. 4 is Western Blot detection BCR-ABL downstream effect developed by molecule figure；
Fig. 5 is Western Blot detection apoptosis pathway figure；
Fig. 6 is colony formation detection cell Proliferation figure.
Embodiment
In order to keep the purpose of the present invention and technical solution clearer, the preferred embodiment of the present invention is carried out below detailed Description.It is noted that following embodiment is served only for that the present invention is further detailed, and should not be understood as to this hair The limitation of bright protection scope.Those skilled in the art's above content according to the present invention make it is some it is nonessential improvement and Adjustment all belongs to the scope of protection of the present invention.The raw materials used in the present invention and reagent are commercial product.Made in following embodiments Experimental method is conventional method unless otherwise specified.The materials, reagents and the like used in the following examples, such as without special Illustrate, is commercially available.
Embodiment 1 designs for people bcr-abl gene specific target site sequence gRNA expression plasmid and constructs (1) according to people The gene order of bcr-abl gene, we utilize 3 pairs gRNA and 1 pair of 4.2 software design of Zifit Targeter Vesion GRNA-half-site synthesizes corresponding oligonucleotides and constructs corresponding gRNA expression plasmid and donor, sequence are shown in Table 1, Corresponding vector plasmid is PSQT1313 (Addgene#53370).The expression plasmid of FokI-dCas9 is PSQT1601 (Addgene#53369)。
The oligo composition sequence of 1 gRNA expression plasmid of table
(2) it synthesizes 3 long segments in raw work to be cloned into PCDH-CMV-MCS-EF1-copGFP carrier, with K562 genome Nearby 338bp is template to DNA aim sequence, is inserted into 8 base NotI restriction enzyme sites (GCGGCCGC), total 346bp.Drawn with upstream The double-strand PCR that object (Forward) SEQ ID NO:7, downstream primer (Reverese) SEQ ID NO:8 amplify target fragment is produced Object, as template donor after using OMEGA kits to recycle.
(3) consideration convey dyeing method
Turn reagent C ell Line Nucleofector Kit V using the electricity that Lonza company provides and nuclear transfection instrument carries out Transfection experiment, concrete operations:
A (contains nuclear transfection solution (Nucleofector Solution) with additive (Supplement) in kit Having) totally 100 μ l are placed in 37 DEG C to 4.5: 1 premixs by volume；
B, 1.5mL culture medium (containing 10%FBS), 37 DEG C of preheatings are added in every hole in 12 orifice plates；
C, every hole count K562 cell 1x106A, 200g is centrifuged 10min；
D, micro sample adding appliance exhaust supernatant, and the nuclear transfection solution preheated in advance is added, and cell is resuspended；
GRNA-17 plasmid 2ug, FokI-dCas9 plasmid 6ug, donor segment is added into cell suspension simultaneously by e 400ng, gently pressure-vaccum mixes；
F opens nuclear transfection instrument, option program T-016；
G is transferred in electric shock tank after the mixing suspension of cell and Plasmid DNA is transferred in electric shock cup, starts to shock by electricity；
H takes out electric shock cup after EP (end of program), the culture medium of 400 μ l preheating is slowly added into along electric shock cup inner wall, rapidly by sample Product are transferred in 12 orifice plates added with culture medium previously preheated.37 DEG C, cultivate 60 hours in 5%CO2 incubator after examined Survey and subsequent experimental.
(4) cell genomic dna extracts:
According to the limited blood/tissue of Tiangeng biochemical technology/cellular genome extracts kit (article No.: DP304) operation step Rapid explanation, the cell genomic dna for the culture 60 hours that extraction step 3 obtains.
(5) target fragment pcr amplification reaction and PCR product sequence verification
The cell genomic dna obtained using above-mentioned steps 4 is template, the upstream primer (Forward) that is used with PCR amplification SEQ ID NO:7, downstream primer (Reverese) SEQ ID NO:8 carry out target fragment pcr amplification reaction.Reaction system is 25 μ l, 12.5 μ l Premix Taq, 1 μ l, 10 μM of primer Sence primer, 1 μ l, 25 μM of primer Anti-sence primer, 100ng genomic DNA adds ultrapure water to 25 μ l.PCR amplification program: 94 DEG C of 5min；94 DEG C of 30s, 56 DEG C of annealing temperature, 72 DEG C 30s is recycled 32 times, last 72 DEG C of extensions 5min.Pcr amplification product is detected with 2% agarose gel electrophoresis.
(6) PCR product carries out sequencing detection
The PCR product that above-mentioned steps obtain directly is sequenced, resulting sequence results and bcr-abl gene will be sequenced In target site sequence carry out tetraploid rice analysis, with identification transfection RFN plasmid K562 cell genomic dna in target site be It is no that NotI restriction enzyme site is inserted by fixed point.Sequencing result peak figure is as shown in Figure 1, the target site of K562 cell genomic dna is determined Point insertion NotI restriction enzyme site.
(7) western blot detects the expression of GAP-associated protein GAP
60 hours K562 cells are cultivated after electricity is turned and cellular control unit is collected respectively, and leach protein carries out western Blot detects the expression of GAP-associated protein GAP.Fig. 2 is Western Blot detection K562 cell BCR-ABL expression figure；Western RFNs+Donor can reduce the expression of BCR-ABL protein in K562 cell to Blot as the result is shown, and the site gRNA-17 effect is the most Obviously.Fig. 3 is Western Blot detection K562 cell γ H2AX expression figure；Western Blot RFNs+Donor as the result is shown K562 γ H2AX can be made to raise, illustrate that double-strand break occurs, the site gRNA-17 effect is the most obvious.It can be with from Fig. 2 and Fig. 3 , it is evident that the site gRNA-17 effect is obvious, BCR-ABL protein is significantly lowered, and γ H2AX is significantly raised, it was demonstrated that site hair Double-strand break is given birth to.Fig. 4 is Western Blot detection BCR-ABL downstream effect developed by molecule figure；Western Blot result Display RFNs+Donor can be such that the activation of K562 and K562/G01 cell downstream effect molecule reduces.Fig. 5 is WesternBlot Detect apoptosis pathway figure；RFNs+Donor can activate the apoptosis of K562 and K562/G01 cell to Western Blot as the result is shown Signal path, and then induce K562 apoptosis.After Fig. 4 and Fig. 5 illustrates that RFNs+donor acts on K562 cell, downstream Effector molecule p-stat5, p-erk, p-crkl activation significantly reduces, which equally exists in K562/G01 cell, explanation RFNs+donor is equally effective to imatinib-resistant cell K562/G01；And detection apoptosis relevant molecule discovery PARP and Caspase3 is activated, and illustrates that Apoptosis has activated caspase access.
(8) flow cytometer detection Apoptosis
GRNA-17+donor, RFNs-half (gRNA-half+FokI-dCas9)+Donor and RFNs+Donor are used respectively Plasmid nuclear transfection K562 and K562/G01 cell after routine culture 60 hours, collect group of cells again, and PBS is carried out after washing twice Annexin V and PE-7AAD dyeing, FCM detect Apoptosis.FCM RFNs+donor is acted on as the result is shown K562 and K562/ G01 Apoptosis obviously increases.
(9) colony formation
GRNA-17+donor, RFNs-half+Donor and RFNs+Donor plasmid nuclear transfection K562 and K562/ are used respectively G01 cell, and set control group (plasmid-free transfection), every group of processing are arranged 4 multiple holes, and every hole spreads 300 cells in 24 orifice plates, Every hole end culture medium is adjusted to 750 μ l, 750 μ l, 2.7% methylcellulose is added in every hole, mixes, cultivates 7-10 days and observes As a result.As a result as shown in fig. 6, colony formation as the result is shown RFNs+Donor can inhibit K562 and K562/G01 cell increase It grows.
Specification sequence table
<110>Medical University Of Chongqing
<120>the gRNA sequence and its application of targeting editor bcr-abl fusion
<160> 8
<210>1
<211> 20
<212>RNA
<213>artificial sequence
<220>
<223>the left subluxation point sequence of gRNA-15
<400>1
gtagcatctg actttgagcc 20
<210>2
<211>20
<212>RNA
<213>artificial sequence
<220>
<223>the right subluxation point sequence of gRNA-15
<400>2
agccgctcgt tggaactcca 20
<210>3
<211> 20
<212>RNA
<213>artificial sequence
<220>
<223>the left subluxation point sequence of gRNA-16
<400>3
ttcagcggcc agtagcatct 20
<210>4
<211>20
<212>RNA
<213>artificial sequence
<220>
<223>the right subluxation point sequence of gRNA-16
<400>4
gtctgagtga agccgctcgt 20
<210>5
<211> 20
<212>RNA
<213>artificial sequence
<220>
<223>the left subluxation point sequence of gRNA-17
<400>5
tttcgttgca ctgtatgatt 20
<210>6
<211>20
<212>RNA
<213>artificial sequence
<220>
<223>the right subluxation point sequence of gRNA-17
<400>6
aacactctaa gcataactaa 20
<210>7
<211> 25
<212>DNA
<213>artificial sequence
<220>
<223>PCR amplification upstream primer
<400>7
tatttttgct tctgagaata aaact 25
<210>8
<211>20
<212>DNA
<213>artificial sequence
<220>
<223>PCR amplification downstream primer
<400>8
caaagggtgg taggtcaaac 20

Claims (8)

1. a kind of gRNA sequence of targeting editor bcr-abl fusion, including gRNA-15, gRNA-16 or/and gRNA-17 sequence Column, the gRNA-15 sequence includes left subluxation point sequence SEQ ID NO:1 and right subluxation point sequence SEQ ID NO:2；It is described GRNA-16 sequence includes left subluxation point sequence SEQ ID NO:3 and right subluxation point sequence SEQ ID NO:4；The gRNA-17 sequence Column include left subluxation point sequence SEQ ID NO:5 and right subluxation point sequence SEQ ID NO:6.
2. gRNA sequence as described in claim 1, it is characterised in that: the sequence is gRNA-17, the gRNA-17 sequence Including left subluxation point sequence SEQ ID NO:5 and right subluxation point sequence SEQ ID NO:6 sequence.
3. the method for targeting editor bcr-abl fusion as claimed in claim 1 or 2, it is characterised in that: by FokI nuclease It is specifically bound to the target site of CML Disease-causing gene bcr-abl, bcr-abl gene is inspired and DNA double chain fracture occurs, and supplying The NotI restriction enzyme site of 8 bases is introduced in body DNA, the bcr-abl of fracture carries out homologous reparation by template of donor dna, makes It causes bcr-abl gene that frameshift mutation occurs after being inserted into 8 bases, so as to cause the destruction of bcr-abl gene.
4. method as claimed in claim 3, it is characterised in that: expressed gRNA, FokI-dCas9 by way of nuclear transfection Carrier and donor are transfected into K562 suspension cell simultaneously, and routine culture 60 hours；By extracting cell genomic dna, PCR Whether the segment of DNA break and homologous recombination occurs for amplification, carry out digestion identification with NotI, detect and be inserted into cellular genome The NotI restriction enzyme site of 8 bases；Then plasmid is transferred to K562 cells play dissection by way of nuclear transfection, destroyed Bcr-abl gene can inhibit BCR-ABL protein to express, and detect GAP-associated protein GAP by Western Blot and Clone formation is real It tests, FCM detects its effect；Upstream primer (Forward) sequence that the PCR amplification uses as shown in SEQ ID NO:7, under Primer (Reverese) sequence is swum as shown in SEQ ID NO:8.
5. a kind of expression vector of the gRNA sequence containing targeting editor's bcr-abl fusion, it is characterised in that: the expression Carrier contains gRNA sequence fragment described in gRNA-15, gRNA-16 or/and gRNA-17.
6. expression vector as claimed in claim 5, it is characterised in that: the expression vector plasmid is PSQT1313.
7. expression vector as claimed in claim 5, it is characterised in that: the expression plasmid of FokI-dCas9 is PSQT1601.
8. application of the gRNA sequence as claimed in claim 1 or 2 in preparation prevention or treatment chronic myelocytic leukemia drug.
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CN106987599A (en) * 2017-03-28 2017-07-28 重庆医科大学 Use Zinc finger nuclease technology to destroy people's bcr abl fusions to suppress CML cells propagation and promote its apoptosis
CN113717991A (en) * 2021-11-01 2021-11-30 菁良基因科技(深圳)有限公司 Method for editing gene fusion

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN106987599A (en) * 2017-03-28 2017-07-28 重庆医科大学 Use Zinc finger nuclease technology to destroy people's bcr abl fusions to suppress CML cells propagation and promote its apoptosis
CN106987599B (en) * 2017-03-28 2021-06-11 重庆医科大学 Zinc finger nuclease for inhibiting expression of human bcr-abl fusion gene or causing loss of function of human bcr-abl gene and application thereof
CN113717991A (en) * 2021-11-01 2021-11-30 菁良基因科技(深圳)有限公司 Method for editing gene fusion
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