CN108893529A - A kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons - Google Patents
A kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons Download PDFInfo
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- CN108893529A CN108893529A CN201810664661.2A CN201810664661A CN108893529A CN 108893529 A CN108893529 A CN 108893529A CN 201810664661 A CN201810664661 A CN 201810664661A CN 108893529 A CN108893529 A CN 108893529A
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- crrna
- kras
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses a kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons, it is characterized in that, it further include that can be with the crRNA in conjunction with CRISPR-Cas13a system, the crRNA Format Series Lines including CRISPR-Cas13a system：5`-Cas13a albumen anchor series-crRNA go-ahead sequence -3`, the advantage of the invention is that, whether the testing result can be mutated by fluorescence reading intuitive judgment people KRAS gene, without high-flux sequence, with it is at low cost, can be repeated several times detection, detection speed it is fast, it the advantages such as a result can intuitively analyze, it is detected suitable for large-scale clinical sample, nucleic acid detection technique disclosed by the invention is different from the previous detection technique of based on PCR technology, and reaction is whole without complicated temperature controller device or system；By being all added three-step reaction in a reaction system, it being further simplified operating process, can detect the nucleic acid fragment with distinguished sequence in two hours.
Description
Technical field
The present invention relates to a kind of detections of biomaterial, specifically, being related to a kind of suitable based on CRISPR-Cas13a technology
Method for quickly detecting nucleic acid specific fragment in biological sample.
Background technique
Disease is studied and diagnosed from gene level, is the pass for realizing accurate this medical medical concept of individuation
Key, Kras are a kind of murine sarcoma virus oncogenes, there are three types of ras gene family genes relevant to human tumor-H-
Ras, K-ras and N-ras are respectively positioned on 11,12 and No. 1 chromosomes.Ras albumen also known as p21 of the K-ras because encoding 21kD
Gene.In ras gene, K-Ras influences maximum to human cancer, it seems molecular switch：It is thin to can control regulation when normal
The path of intracellular growth；When being abnormal, then lead to cell continued propagation, and prevents cell self-destruction.It participates in intracellular
Signal transmitting, when K-ras gene mutation, which is permanently activated, and cannot be generated normal ras albumen, be made Intracellular signals
Conduction disturbance, uncontrolled cellular proliferation and canceration.
Conventional genetic test is realized by round pcr at present, but there are such as sensitivity of some defects for existing round pcr
Not high, specific not strong, target fragment causes at high cost, repeated not strong, detection time-consuming to wait so long problem frequently with DNA etc..
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of detection people KRAS gene 2
And 3 exon mutation crRNA.
A kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons, including
CRISPR-Cas13a system further includes that can be with the crRNA in conjunction with CRISPR-Cas13a system, the crRNA Format Series Lines：
5`-Cas13a albumen anchor series-crRNA go-ahead sequence -3`.
The Cas13a albumen is Lw Cas13a, and the anchor series in the crRNA sequence are
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC,
The crRNA sequence is respectively
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCC AACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUG UGUCGA；
The picodna sequence for transcribing crRNA is
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAA
TCCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAA
ATCCCCTATAGTGAGTCGTATTAATTTC。
The Cas13a albumen is Lsh Cas13a, and the anchor series in crRNA sequence are
CCACCCCAAUAUCGAAGGGGACUAAAAC,
The crRNA sequence is respectively
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA is for transcribing crRNA
Picodna sequence be
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTAT
AGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTAT
AGTGAGTCGTATTAATTTC。
A kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons
Application in people KRAS gene extron 2 and exon 3 abrupt climatic change.
A kind of nucleic acid detection technique based on CRIPSR-Cas13a disclosed by the invention, most important mechanism are
Cas13a albumen can be identified with the help of guide RNA with targeting sequence RNA segment, be subsequently activated not by sequence
The RNA enzymatic activity of limitation, by the way that signal reports molecule caused by RNA chain degradation is added in the reaction system, final realize has
Target the signal identification of the RNA segment of sequence.
The present invention acts on the lower RNA synthesized by t7 rna polymerase, is not only expanded to the application range of the detection technique
RNA and DNA can be detected, while significantly improve the sensitivity of detection, can detecte out the nucleic acid of A Moer rank.
Since Cas13a albumen is when identification targets sequence, it can be usually resistant to the mispairing of a base, the present invention passes through
Artificial base mismatch is added in the sequence of guide RNA in advance, reaching the specificity of this detection technique can identify there is list
The nucleic acid sequence of base difference.
Template of the present invention, which can be DNA, in contrast can also be RNA, and testing result can pass through fluorescence reading intuitive judgment people
Whether KRAS gene mutates, and without high-flux sequence, has that at low cost, can be repeated several times detection, detection speed fast,
It the advantages such as a result can intuitively analyze, be suitable for large-scale clinical sample and detect.
Nucleic acid detection technique disclosed by the invention is different from the previous detection technique of based on PCR technology, reacts whole nothing
Need complicated temperature controller device or system；By being all added three-step reaction in a reaction system, it is further simplified
Operating process can detect the nucleic acid fragment with distinguished sequence in two hours.
Specific embodiment
By the technology contents that the present invention will be described in detail, construction feature, reached purpose and efficacy, hereby enumerates embodiment below
It is explained in detail.
A kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons, including
CRISPR-Cas13a system further includes that can be with the crRNA in conjunction with CRISPR-Cas13a system, the crRNA Format Series Lines：
5`-Cas13a albumen anchor series-crRNA go-ahead sequence -3`；The Cas13a albumen can be Lw Cas13a, described
Cas13a albumen also can be Lsh Cas13a.
When Cas13a albumen is Lw Cas13a
Anchor series in crRNA sequence are crRNA described in GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC
Sequence is respectively
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGC UCCAACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUG CUGUGUCGA；
The picodna sequence for transcribing crRNA is
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCT
AAATCCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTC
TAAATCCCCTATAGTGAGTCGTATTAATTTC。
When the Cas13a albumen is Lsh Cas13a
Anchor series in crRNA sequence are CCACCCCAAUAUCGAAGGGGACUAAAAC,
The crRNA sequence is respectively
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA
Picodna sequence for transcribing crRNA is
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCC
TATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCC
TATAGTGAGTCGTATTAATTTC。
Target the crRNA design principle of people KRAS gene
Since CRISPR-Cas13a system is a kind of novel targeted rna gene editing system.Wherein Cas13a is a kind of
Double-core ribonuclease T., Cas13a form monitoring compound in conjunction with crRNA, and the guide region of crRNA identifies mesh with complementary series
Mark RNA, and Cas13a degradation target RNA chain.At present due to not specific crRNA design principle, according to previous work and
Experience, the crRNA design requirement for targeting people KRAS gene are as follows：
CrRNA includes albumen anchor series and go-ahead sequence, and Format Series Lines are：5`- and the protein bound anchoring of Cas13a
Sequence-crRNA go-ahead sequence -3`, albumen anchor series needs determined according to Cas13a albumen, can with it is selected
Cas13a protein matches simultaneously combine；Go-ahead sequence then with targeting DNA in fragment match.
The selection of guide crRNA RNA
People KRAS gene order is searched according to NCBI, determines the site that exon 2 and exon 3 mutate；crRNA
Target spot cannot be too close from initiation codon (ATG)；Length is 21-28 nucleotide；Finally, targeting people KRAS respectively is designed
The crRNA go-ahead sequence of gene extron 2 and exon 3, the target site DNA sequence dna being directed to is as shown in table 1, wherein capitalization portion
It is divided into the codon that can be mutated.
The mutational site target sequence of people's KRAS gene
With the design of the protein bound anchor series of Cas13a：
When Cas13a albumen selects Lw Cas13a, the anchor series in crRNA sequence are
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC
When Cas13a albumen selects Lsh Cas13a, the anchor series in crRNA sequence are
CCACCCCAAUAUCGAAGGGGACUAAAAC
The synthesis of crRNA
T7 promoter sequence, the T7 promoter sequence are added by chemically synthesized crRNA reverse transcription at DNA and in 5`
For 5`-TAATACGACTCACTATAGGG-3`；Above-mentioned band T7 promoter DNA is generated into RNA under t7 rna polymerase effect, is returned
Purifying is received, enough crRNA are obtained, can also be polymerize according to the crRNA transcription templates DNA with T7 promoter of design in T7RNA
RNA is generated under enzyme effect, recovery purifying obtains enough crRNA.
RNA, the corresponding Cas13a egg that the crRNA of the targeting people KRAS gene of synthesis, sample DNA transcription are generated
White, RNA reporter (RNAse Alert V2, Thermo Scientfic) in nuclease buffer, 37 DEG C be incubated for 1~
3 hours, KRAS gene extron 2 and exon 3 catastrophe can intuitively be analyzed by fluorescence reading.It is described based on
CRISPR technology specific detection people KRAS gene 2 and the crRNA of 3 exons mutation can be applied to people's KRAS gene
The detection of exon 2 and exon 3 mutation.
In specific implementation, selects KRAS wild-type gene fragment and KRAS mutation type genetic fragment as detection model, press
Two sections of genes are had detected according to the nucleic acid detection technique of Invention Announce, can detecte out the KRAS wild-type gene fragment of about 50aM,
The design for passing through guide RNA simultaneously, can distinguish the DNA nucleic acid sequence an of base difference, i.e. KRAS wild type gene piece
Section and KRAS-G12D mutated genes segment.
In conclusion only the preferred embodiments of the invention, is not limited the scope of protection of the present invention with this, it is all according to the present invention
Equivalent changes and modifications made by the scope of the patents and description are all within the scope of the invention patent covers.
Claims (4)
1. a kind of crRNA being mutated based on CRISPR technology specific detection people KRAS gene 2 and 3 exons, feature
It is, including CRISPR-Cas13a system, further including can be with the crRNA in conjunction with CRISPR-Cas13a system, the crRNA sequence
Column format is：5`-Cas13a albumen anchor series-crRNA go-ahead sequence -3`.
2. according to claim 1 a kind of based on CRISPR technology specific detection people KRAS gene 2 and 3 exons
The crRNA of mutation, it is characterised in that：The Cas13a albumen is LwCas13a, and the anchor series in the crRNA sequence are
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC, the crRNA sequence are respectively
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA；
The picodna sequence for transcribing crRNA is
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGA
GTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGA
GTCGTATTAATTTC。
3. according to claim 1 a kind of based on CRISPR technology specific detection people KRAS gene 2 and 3 exons
The crRNA of mutation, it is characterised in that：The Cas13a albumen is LshCas13a, and the anchor series in crRNA sequence are
CCACCCCAAUAUCGAAGGGGACUAAAAC, the crRNA sequence are respectively
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA
Picodna sequence for transcribing crRNA is
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATT
AATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATT
AATTTC。
4. one kind described in claim 1-3 is based on CRISPR technology specific detection people KRAS gene 2 and 3 exons are prominent
Application of the crRNA of change in people KRAS gene extron 2 and exon 3 abrupt climatic change.
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| 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 |
| CN115404268A (en) * | 2022-10-11 | 2022-11-29 | 芜湖森爱驰生物科技有限公司 | SRY gene detection probe and kit |
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