CN109897852A - The gRNA of tumour related mutation gene based on C2c2, detection method, detection kit - Google Patents
The gRNA of tumour related mutation gene based on C2c2, detection method, detection kit Download PDFInfo
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Abstract
The present invention provides a kind of gRNA for target tumor related mutation gene RNA, and the present invention also provides a kind of human tumour related mutation gene testers based on short palindrome repetitive sequence (the CRISPR)-C2c2 system of regular intervals cluster, detection kit.The present invention provides detection methods, combine the advantage of gRNA targets identification tumour related mutation gene transcript RNA (target RNA sequence) and when CRISPR-C2c2 compound detects target RNA sequence, compound can cut the reporter rna with detection label, the characteristics of discharging detectable signal, CRISPR-C2c2 system is applied in tumour related mutation genetic test, high sensitivity, accuracy are high, are a kind of detection methods and detection kit with huge commercial application value.
Description
Technical field
The present invention relates to genetic test and gene modification fields, are related to a kind of for selectively targeted tumour related mutation base
Because of the gRNA of RNA, and a kind of tumour related mutation base for being based on short palindrome repetitive sequence (CRISPR) system of regular intervals cluster
Because of detection method, detection kit.
Background technique
The regular short palindrome in cluster interval repeats system (clustered regularly interspaced short
palindromic repeat；CRISPR-associated, CRISPR-Cas) be archaeal and bacterium resistance virus and plasmid
The important immune defense system infected, for resisting the invasion of exogenous genetic material, such as phage virus and exogenous plasmid.Together
When, it provides acquired immunity for bacterium: this is similar with the acquired immunity of mammal, when bacterium is by viral or outer
When source plasmid is invaded, corresponding " memory " can be generated, so as to resist their invasion again.CRISPR/Cas system can be with
It identifies exogenous DNA or RNA, and they is cut off, the expression of silencing foreign gene.Just because of this accurate targeting function
Can, CRISPR/Cas system is developed to a kind of efficient gene editing tool.
CRISPR-Cas system is divided into two major classes, and first major class CRISPR-Cas system is answered by the multi-subunit effect formed
Object is closed to function；Second major class is functioned by single effect protein (such as Cas9, Cpf1, C2c1 etc.).Wherein,
Cas9, Cpf1, C2c1 all have the DNA endonuclease activity of RNA mediation.Currently, Cas9 and Cpf1 albumen is as genome
Edit tool is widely used, and overcomes the disadvantages of traditional gene editing technical step is cumbersome, time-consuming, low efficiency, with its compared with
Few ingredient, easily operation and higher efficiency meet the gene editing demand in most of fields, and have it is potential and
Huge clinical value.
In nature, CRISPR/Cas system possesses plurality of classes, wherein CRISPR/Cas9 system be study it is most deep
Enter, a kind of most mature classification of application.CRISPR-Cas9 is a kind of complex with endonuclease activity, is identified specific
DNA sequence dna, carry out specific site cutting causes double-strand DNA cleavage (Double-strand breaks, DSB), in no mould
Under conditions of plate, non-homogeneous recombination end connection (Non-homologous end joining, NHEJ) occurs, causes frameshit
It is mutated (frameshift mutation), leads to gene knockout.CRISPR/Cas9 be after " zinc finger endonuclease (ZFN) ",
The third generation " genome fixed point editing technique " occurred after " class activating transcription factor effector nuclease (TALEN) ".Rely on
It is low in cost, it is easy to operate, it is high-efficient the advantages that, the worldwide laboratory rapidly CRISPR/Cas9 becomes biological section
The strong helper ground.
CRISPR/Cas is the strong tools for carrying out gene editing, can be to the accurate edits that gene is pinpointed.To
Lead under RNA (guide RNA, gRNA) and the participation of Cas9 albumen, cell genomic dna to be edited will be counted as virus or
Exogenous DNA is accurately sheared.But there are also restrictive conditions for the application of CRISPR/Cas9.Firstly, region to be edited
The PAM sequence (NGG) for nearby needing to have relatively conservative.Secondly, guide RNA will be with the series complementary pairing of the upstream PAM.
If respectively designing a guide RNA (guide RNA1, guide RNA2) in the upstream and downstream of gene, it is compiled with containing Cas9 albumen
The plasmid of code gene is transferred in cell together, and guide RNA can target the target sequence near PAM by base pair complementarity,
Cas9 albumen can be such that the DNA double chain of the gene upstream and downstream is broken.And there is the answering machines of DNA damage reparation for organism itself
System can connect the sequence for being broken upstream and downstream both ends, to realize the knockout of target gene in cell.If in this base
The template plasmid (donor DNA molecule) of a reparation is introduced on plinth for cell, this like cell will repaired according to the template of offer
Segment insertion or rite-directed mutagenesis are introduced during multiple.Gene editing is carried out to fertilized egg cell, and is conducted into foster mothers,
The building of gene editing animal model may be implemented.With going deep into for research, CRISPR/Cas technology has been widely used.
In addition to gene knockout, the basis such as gene replacement edit mode, it may be utilized for gene activation, and disease model constructs, even
It is gene therapy.
It is by two kinds of tiny RNAs that Cas9, which targets cutting DNA, --- gRNA (CRISPR RNA) and tragRNA (trans-
Activating gRNA) and target sequence complementation identification principle realize.Two kinds of tiny RNAs one has been fused into now
RNA chain, abbreviation gRNA (guide RNA).Therefore, can gRNA 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 CRISPR-
Specific knockdown of the Cas9 to target gene.Therefore, it can design, prepare accuracy and selectively targeted target gene
GRNA becomes the key technology of CRISPR-Cas9 gene knockout.
2015, a kind of the second completely new class CRISPR-Cas system-VI type system was found, the effect egg in the system
It is white to be named as C2c2.Entitled " the Discovery that Zhang Feng team delivers on November 5th, 2015 at " Mol Cell "
The article of 2 CRISPR-Cas Systems " of and Functional Characterization of Diverse Class.
According to article it is found that the breakthrough meaning of this system is that it can edit target RNA, rather than traditional DNA
Editor.The basic workflow of the system is similar with CRISPR/Cas9, or by " blacklist " system of CRISPR sequence
System hits invader.But the mode that gRNA is formed is different from CRISPR/Cas9 system: C2c2 albumen can be with maturation
GRNA is compound, and in the case where not by tragRNA in conjunction with external source single stranded RNA, gRNA then can be with PFS segment (similar PAM)
Neighbouring complementary region hybridization.Finally, external source single stranded RNA can be sheared, gene expression can be also silenced.However, cutting
While target RNA, the C2c2 having activated can also degrade target RNA neighbouring RNA, referred to as " attached cutting ".This targeting is made
For RNA and assist the ability for executing genome instruction that can allow people specifically and with high throughput manipulation or labeled RNA,
And more widely operator function.Research then is it has furthermore been found that VI type CRISPR-Cas system is a kind of novel
The CRISPR system of targeted rna, and C2c2 be it is a kind of with RNA be guiding targeting and degradation of rna endonuclease, be expected to by
The tool studied as RNA is developed, utilization of the CRISPR system in terms of gene editing is extended.
On October 13rd, 2016, entitled " the Two distinct RNase that Doudna team delivers at " Nature "
The opinion of activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection "
Literary and on January 12nd, 2017 delivers the entitled of the gorgeous seminar of king, biophysics research institute, the de Chinese Academy of Sciences in Cell magazine
The research of " Two Distant Catalytic Sites Are Responsible fbr C2c2 RNase Activities "
All show gRNAhC2c2 and play its two different RNA enzyme by two independent active structure domains to cut activity, this is research
C2c2 plays the active molecular mechanism of RNA enzyme and provides important structure biology basis.
Wang Yanli seminar by in-depth study, parsed the binary complex of C2c2 and gRNA crystal structure and
The crystal structure of C2c2 albumen discloses structural domain i.e. REC structural domain and a nuclease that C2c2 includes gRNA identification
Structural domain, that is, NUC structural domain.REC structural domain includes N-terminal structural domain (N-terminal domain) and Helical-1 structure
Domain, NUC structural domain contain two HEPN structural domains, Helical-2 structural domain and the connection for connecting two HEPN structural domains
Structural domain.The active region for being responsible for cutting precursor gRNA and target RNA is located on Helical-1 and HEPN structural domain.gRNA
Combination can cause the conformation change of C2c2 albumen, this variation is likely to stablize the combination of gRNA, and then to activation C2c2
The activity of target RNA is cut, degrade target RNA and other RNA by near target RNA.The above research is disclosed by structure and Biochemical Research
C2c2 shearing pre-gRNA and the molecular mechanism for cutting target RNA, the molecule base of RNA virus invasion is resisted to understanding bacterium
Plinth has a very important significance.Also it is simultaneously transformation CRISPR-C2c2 system, is provided for its utilization in gene editing field
Strong structure basis helps speed up the understanding of the disease caused to virus infection, treats and prevents.In addition,
The research of Doudna professor also found, can use the effect set reporter rna of such " the attached cutting " of C2c2 to detect target
The presence or absence of RNA.
On April 13rd, 2017, Science magazine deliver an entitled " Nucleic acid detection with
The important research of CRISPR-Cas13a (i.e. C2c2) " is in progress.One from mechanisms such as Broad research institute, McGovern research institutes
The CRISPR system of targeted rna has been transformed in a scientist group, has become quick, cheap and super-sensitive diagnosis work
Tool.This discovery is expected to bring the influence for the property changed for scientific research and global public health.CRISPR pioneer Zhang Feng and
The James J.Collins of Broad research institute is the common communication author of the research.The graduate researcher of Broad refers to
Out, a kind of new CRISPR technology: CRJSPR-Cas13a/C2c2 is utilized, highly sensitive can detect includes that zika virus infects,
Disease including dengue virus infection etc., principle are that by CRISPR-Cas13a in conjunction with isothermal nucleic acid amplification, detection is special
Anisotropic RNA and DNA.
Researcher points out that Cas13a has unique property, Cas13a and DNA target to CRISPR enzyme (such as Cas9 and Cpf1)
Difference, this enzyme can keep activity after cutting its targeted rna, and may show to mix behavior (promiscuous
Behavior), continue to cut other non-targeted RNA, this is known as " attached cutting " (collateral by the laboratory Zhang Feng
Cleavage, biology interpreter).This activity is detection of nucleic acids platform SHERLOCK (Specific High newly developed
Sensitivity Enzymatic Reporter UnLOCKing) key feature, in addition, SHERLOCK also includes one
The reporter rna of fluorescence is issued when a cutting.When Cas13a detects target RNA sequence, RNase activity will cut report
RNA discharges detectable fluorescence signal." it is understood that Cas13a has sensitive attached activity, but initially we analyze its spy
When sign, its sensitivity is found not enough, " the other postgraduate in the laboratory Zhang Feng: Jonathan Gootenberg is said.In order to
This problem is solved, which cooperates with James Collins, using the papery stockaded village card disease researched and developed before Collin study group
Malicious detection technique.The new system that both technologies are combined can detect list RNA and single DNA molecular with extremely low concentration.
Tumour is the current maximum disease for threatening human health, alreadys exceed coronary heart disease, headstroke in Chinese tumour, becomes
First cause of the death.Targeted drug is directed to tumour-specific site, by locally maintaining higher concentration, improves for tumour
Specific killing power, normal tissue cytosis is smaller, avoids the side effect of chemotherapeutic treatment.
Targeted drug and oncogene are mutated there are relevance, before selecting targeted drug treatment, need to confirm tumour
Gene mutation site, for instructing, selecting targeted drug, and whether precognition drug effective.Cancer target traditional at present
Drug related mutation gene tester has Sanger PCR sequencing PCR, fluorescence quantitative PCR method, Fish method, micro array etc..It passes
For the detection method of gene mutation of system because technology single can only detect less mutational site, sensitivity is low.
In Sanger PCR sequencing PCR, single pair primer can detect multiple mutation, but outer to the difference of multiple genes or same gene
Mutational site on aobvious son just needs to carry out amplification sequencing respectively, cumbersome, and sensitivity lower about 20%, false negative rate compared with
It is high.Although fluorescence quantitative PCR method high sensitivity, each pair of primer can only detect a kind of mutation, and every kind of mutation need to be established individually
One PCR reaction system, while it is cumbersome to detect the multiple mutational sites of multiple samples.Requirement of the both methods to sample
It is all larger, be not suitable for while detecting multiple gene mutation sites.
Applicants have found that not there is the tumour related mutation genetic test side based on CRISPR-C2c2 system also at present
The relevant report of method.GRNA as designing, preparing accurate, selectively targeted target gene is CRISPR-Cas9 clpp gene
The key technology removed, the gRNA of efficient, selectively targeted target tumor related mutation gene are also CRISPR-C2c2 identification
The key of target gene, and further such that the editor of the tumour related mutation gene based on CRISPR-C2c2 system, repair
Decorations, detection are possibly realized.
Therefore, intend applying for a kind of tumour related mutation gene tester based on CRISPR-C2c2 system, detection reagent
Box and gRNA for selectively targeted tumour related mutation gene RNA.
Any manufacturer of any product in referenced herein or any document for being incorporated herein
Specification, explanation, product specification and product table, be incorporated herein by reference, and can adopt in the practice of the invention
With.More specifically, the document of all references is both incorporated herein by reference, and degree is as definite in each individual document
Ground and individually indicating is incorporated by reference into herein.
Summary of the invention
To solve the above problems, the present invention provides a kind of for selectively targeted tumour related mutation gene RNA
GRNA and the tumour related mutation gene tester based on CRISPR-C2c2 system, detection kit.
If technical solutions according to the invention preferably use CRISPR-C2c2 system without specified otherwise.
First aspect present invention provides a kind of gRNA sequence, and the target nucleotide is that tumour related mutation gene pairs is answered
RNA sequence, the corresponding DNA sequences encoding of the gRNA includes core shown in SEQ ID NO.1-SEQ ID NO.306 in table 3
One or more in nucleotide sequence.
In one embodiment of the invention, the tumour related mutation gene is that pathogen-resistance gene and/or pathogen are special
Gene.
In one embodiment of the invention, the tumour related mutation gene includes swelling shown in gene number 1-83 in Tables 1 and 2
Tumor related mutation gene it is one or more.In one embodiment of the invention, " the corresponding RNA sequence of tumour related mutation gene
Column " are the corresponding transcript of tumour related mutation gene.
In embodiment of the present invention, term " the corresponding DNA sequences encoding of gRNA " obtains the present invention the after transcription
On the one hand the gRNA sequence specifically can be used and the corresponding DNA sequences encoding of gRNA is cloned into comprising T7 promoter
In carrier, or by modes such as PCR, annealing, synthesis directly in the front end of the corresponding coding DNA of gRNA plus T7 promoter, warp
It transcribes and can get transcription product gRNA sequence.
It is worth noting that, the applicant is equal for tumour related mutation gene shown in gene number 1-83 in Tables 1 and 2
Devise a plurality of gRNA, at most design gRNA1-4 totally four gRNA, every gRNA can independently, the specific recognition row pair
The tumour related mutation gene answered.
For example, the tumour related mutation gene is BRCA1, the corresponding DNA sequences encoding of gRNA includes SEQ ID in table 3
One or more in nucleotide sequence shown in NO.1-SEQ ID NO.4.The tumour related mutation gene is BRCA2, gRNA
Corresponding DNA sequences encoding includes one or more in nucleotide sequence shown in SEQ ID NO5-SEQ ID NO.8 in table 3.
And so on, it will be appreciated by persons skilled in the art that each pathogen shown in gene coding 1-83 in Tables 1 and 2
Gene respectively corresponds one or more gRNA in table 3.Applicant does not illustrate one by one.
Base abbreviation annotation of the present invention (it is understood that not annotating letter if any other, makees this field as follows
It is conventional to understand):
H is a, c or t,
B is g or c or t,
D is a or g or t,
V is a or g or c,
N is a or g or c or t.
In one embodiment of the invention, gRNA can be by complete complementary or substantially complementary or special with certain complementing percentage
Property the identification target nucleotide sequences.
GRNA provided by the present invention and the target nucleotide sequences have it is complementary enough so as to the target nucleotide sequence
Column hybridize and instruct the specific binding of CRISPR-C2c2 Yu the target nucleotide sequences.In some embodiments, gRNA with
Complementarity between its corresponding target nucleotide sequences be about or more than about 50%, 60%, 75%, 80%, 85%, 90%,
95%, 97.5%, 99% or more.
In the embodiment of the present invention, " complementation " refers to nucleic acid and another nucleic acid sequence by means of traditional Watson-Crick
Base pairing or other non-traditional types form one or more hydrogen bonds." complementing percentage " indicates can be in a nucleic acid molecules
One second nucleotide sequence formed the residue of hydrogen bond (for example, Watson-Crick base pairing) percentage (for example, 10 it
In to have 5,6,7,8,9,10 be 50%, 60%, 70%, 80%, 90% and 100% complementary)." complete complementary " indicates one
All consecutive residues of a nucleic acid sequence form hydrogen bond with equal number of consecutive residue in a second nucleotide sequence.Such as this
" being substantially complementary " that text uses refer to one have 8,9,10,11,12,13,14,15,16,17,18,19,20,21,
22, on 23,24,25,30,35,40,45,50 or more the regions of nucleotide be at least 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% complementarity.
In an embodiment of the present invention, the gRNA is to target sequence to connect the mature gRNA of composition with gRNA frame sequence
Sequence.
In an embodiment of the present invention, the corresponding DNA sequences encoding of the targeting sequence is SEQ ID NO.1- in table 3
One or more in nucleotide sequence shown in SEQ ID NO.306.
Common gRNA frame sequence includes but is not limited to:
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC。
In the case where not by tracrRNA, C2c2 albumen (i.e. Cas13a) can be compound with gRNA, forms Cas13a-
GRNA compound, when Cas13a-gRNA compound detects target nucleotide (present invention is denoted as first object nucleic acid),
RNase activity will be broken, cut or marker target nucleotide and reporter rna (present invention is denoted as the second target nucleic acid, the report
RNA is the tool of the subsequent second aspect of the present invention, " sequence relevant to target nucleotide " described in the third aspect and fourth aspect
One kind of body embodiment)；Wherein, first object nucleic acid, can be compound by Cas13a-gRNA with PFS segment (similar PAM)
GRNA specific recognition in body.
It can be the reporter rna chain with fluorescent marker, reporter rna chain by the second target nucleic acid in an embodiment of the present invention
After being sheared, fluorescence can be issued, therefore, can detect by detection fluorescence, judge first object nucleotide.
In an embodiment of the present invention, Cas13a-gRNA complex is specific to the identification of first object nucleic acid.
In an embodiment of the present invention, Cas13a-gRNA complex is nonspecific to the cutting of the second target nucleic acid.
In embodiment of the present invention, " the Cas13a-gRNA complex/object ", " CRISPR-Cas13a complex/object ",
" CRISPR-C2c2 complex/object " concept can be interchanged.
In embodiments of the present invention, term " target nucleotide ", " first object nucleic acid ", " the second target nucleic acid " refer to core
Ribotide or its analog.It is the non-limiting of " target nucleotide ", " first object nucleic acid " or " the second target nucleic acid " below
Example: mRNA (mRNA), transfer RNA, rRNA, short interfering rna (siRNA), short hairpin RNA (shRNA), micro-
RNA (miRNA), ssRNA or any isolated RNA (specifically, including single stranded RNA or with the double-stranded RNA of single stranded RNA)." target
Nucleotide ", " first object nucleic acid ", " the second target nucleic acid " may include one or more modified nucleotide, such as methyl
The nucleotide and nucleotide analog of change.Nucleotide can pass through molecular labeling (such as fluorescent marker or other detectable molecules
Label) further modify.
In embodiments of the present invention, term " target nucleotide " and " target polynucleotide " can be interchanged.
Second aspect of the present invention provides a kind of CRISPR-C2c2 system, comprising:
1) C2c2 effect protein；
2) one or more nucleic acid, wherein one or more nucleic acid include it is at least one as described in relation to the first aspect
GRNA sequence；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 is compound
When object is in conjunction with target nucleotide described in first aspect, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide is modified.
In a preferred embodiment of this invention, the modification is to introduce fracture, cutting or label.
In one embodiment of second aspect of the present invention, the target nucleotide includes target nucleotide (target described in first aspect
) and/or reporter rna RNA.
Third aspect present invention provides a kind of non-naturally occurring or engineering composition, and the composition includes one
A or multiple carriers, the one or more carrier include component I and component II:
The component I includes the first regulating element, and the coding being operably connected with first regulating element
The coded sequence of C2c2 albumen；The component II includes the second regulating element, and operationally with second regulating element
The coded sequence of the coding gRNA of connection, wherein the gRNA includes gRNA sequence as described in relation to the first aspect；
Wherein, component I and II is located on identical or different carrier；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 is compound
When object is in conjunction with target nucleotide described in first aspect, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide is modified.
In a preferred embodiment of this invention, the modification is to introduce fracture, cutting or label.
In one embodiment of third aspect present invention, the target nucleotide includes target nucleotide (target described in first aspect
) and/or reporter rna RNA.
In embodiments of the present invention, term " carrier " refers to a kind of nucleic acid molecules, it can transport connected to it another
A kind of nucleic acid molecules.Carrier includes but is not limited to single-stranded, double-strand or partially double stranded nucleic acid molecules；Certainly including one or more
By end, the nucleic acid molecules without free end (such as cricoid)；Nucleic acid molecules including DNA, RNA, or both；And this field
Other known diversified polynucleotides.Optionally, a type of carrier is " plasmid ", and referring to wherein can be such as
The circular double stranded DNA ring of other DNA fragmentation is inserted by standard molecule clone technology.Optionally, another type of carrier
It is viral vectors, wherein DNA derived from virus or RNA sequence are present in for packaging virus (for example, retrovirus, duplication
Deficiency retrovirus, adenovirus, replication-defective adenoviral and adeno-associated virus) carrier in.Viral vectors also wraps
Containing the polynucleotides carried by the virus for being transfected into a kind of host cell.Certain carriers are (for example, have bacterium to replicate
The bacteria carrier and episomal mammalian vectors of point) it can independently be replicated in their imported host cells.Other are carried
Body (for example, non-add type mammalian vector) is integrated into the genome of the host cell after being introduced into host cell, and
Thus it is replicated together with the host genome.Moreover, the expression for the gene that certain carriers can instruct them to be operatively connected.This
The carrier of sample is referred to herein as " expression vector ".It is usually plasmid form that carrying agent, which is commonly expressed, used in the recombinant DNA technology.
In general, " being operably connected " is intended to indicate that nucleotide sequence allows the nucleotide sequence with a kind of in carrier
The mode of expression be connected to one or more regulating elements (optionally, carrier be in a kind of in-vitro transcription/translation system
The nucleotide sequence can be expressed；Optionally, the nucleotides sequence can be expressed when the carrier is introduced in host cell
Column).
In embodiments of the present invention, term " expression " refers to that being transcribed into polynucleotides from DNA profiling (is such as transcribed into mRNA
Or other RNA transcripts) process and/or the mRNA of transcription then translate into the process of peptide, polypeptide or protein.Transcript and
The polypeptide of coding can collectively referred to as " gene product ".If polynucleotides derive from genomic DNA, expression may include that eukaryon is thin
The montage of mRNA in born of the same parents.
Terms used herein " non-naturally occurring " or " engineering " are interchangeably used, when referring to nucleic acid molecules or more
When peptide, indicate that the nucleic acid molecules or polypeptide are found in tying in nature with it at least substantially from them in nature or such as
At least another component separate out closed.
In a preferred embodiment of this invention, first regulating element includes one or more pol III promoter
(such as 1,2,3,4,5, or more pol III promoter), one or more pol II promoter (such as 1,2,3,4,5 or
More pol II promoters), one or more pol I promoter (such as 1,2,3,4,5, or more pol I starting
Son), or combinations thereof.The example of pol III promoter includes but is not limited to U6 and H1 promoter.The example of pol II promoter
Including but not limited to reverse transcription Rous sarcoma virus (RSV) LTR promoter (optionally there is RSV enhancer), cytomegalovirus
(CMV) promoter (optionally having cmv enhancer) is [see, e.g., wave Saudi Arabia (Boshart) et al., " cell " (Cell)
41:521-530 (1985)], SV40 promoter, dihyrofolate reductase promoter, beta-actin promoter, phosphoglycerol swash
Enzyme (PGK) promoter and EF1 α promoter.
In some embodiments of the present invention, the coded sequence of C2c2 albumen is encoded through codon optimization, so as to specific
Cell such as eukaryocyte in express.These eukaryocytes can be those of particular organisms or from particular organisms, such as feed
Newborn animal, including but not limited to people, mouse, rat, rabbit, dog or non-human primate.In general, codon optimization is
Refer to and native sequences are replaced at least by the codon more frequently or most frequently used in the gene of host cell
One codon (for example, about or more than about 1,2,3,4,5,10,15,20,25,50, or more codon maintain simultaneously should
Natural acid sequence and modify a nucleic acid sequence to enhance the method for the expression in host cell interested.Different
Species show specific preference for certain codons with specific amino acids.Codon preference is (between biology
The difference that codon uses) it is often related to the translation efficiency of mRNA (mRNA), and the translation efficiency is then considered depending on
(except other things) availability of the property for the codon being translated and specific transfer RNA (tRNA) molecule.It is intracellular selected
The advantage of tRNA generally reflect the codon for being used most frequently for peptide synthesis.Therefore, gene can be customized to be based on password
Best gene expression of the son optimization in given biology.Codon usage table can be readily available, such as be made in codon
With in database (" Codon Usage Database "), and these tables can be adjusted by different modes it is applicable.Referring to,
Middle village Y. (Nakamura Y.) et al., " codon tabulated from international DNA sequence data library uses: state in 2000 "
(Codon usage tabulated from the international DNA sequence databases:status
For the year 2000) " nucleic acids research " (Nucl.Acids Res.) 28:292 (2000).For codon optimization spy
Fixed ordered series of numbers so as to the computerized algorithm expressed in specific host cell be also it is available, as gene manufacture (Gene
Forge) (Aptagen company；Chris Jacobs (Jacobus), PA) and it is available.In some embodiments, it is encoding
In the sequence of CRISPR enzyme one or more codons (such as 1,2,3,4,5,10,15,20,25,50, or more,
Or all codons) correspond to the codon most frequently used for specific amino acids.
In embodiments of the present invention, term " C2c2 ", " C2c2 albumen ", " C2c2 effect protein ", " Cas13a ",
" Cas13a albumen ", " Cas13a effect protein " can be interchanged；C2c2 albumen is the RNase of RNA- targeting, cuts ssRNA
(single-stranded microRNA).The equally applicable embodiment of the present invention of C2c2 albumen disclosed in existing literature, the existing literature include but
It is not limited to:
1、Discovery and Functional Characterization of Diverse Class 2
CRISPR-Cas Systems, Nature, 2016 Oct 13；
2、C2c2 is a single-component programmable RNA-guided RNA-targeting
CRISPR efiector, Science, 2016 Aug 5；
3、Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA
Processing and RNA detection, Nature, 2016 Oct 13；
4、Two Distant Catalytic Sites Are Responsible fbr C2c2 RNase
Activities, Cell, 2017 Jan 12.
In an embodiment of the present invention, C2c2 albumen be from Leptotrichia wadei F0279 or
The Cas13a protein gene of Leptotrichia shahii, i.e. respectively LwCas13a, LshCas13a.
In an embodiment of the present invention, C2c2 albumen includes the homologue or its modified forms of C2c2 albumen.
In an embodiment of the present invention, the component I further includes the coding of any other protein or polypeptide domain
Sequence, and the catenation sequence optionally between any two structural domain, the catenation sequence specifically, codified such as C2c2
The joining peptide of albumen and any other protein or polypeptide domain, and obtain fusion protein.C2c2 albumen can be fused to
On protein domain example include but is not limited to epitope tag, reporter sequences and have following active one
The protein domain of person or more persons: methyl enzymatic activity, hepatic Microsomal Aniline Hydroxylase, transcriptional activation activity, transcription repression activity, transcription
Releasing factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.The non-limiting example of epitope tag
Including histidine (His) label, V5 label, FLAG label, influenza virus hemagglutinin (HA) label, Myc label, VSV-G mark
Label and thioredoxin (Trx) label.The example of reporter gene include, but are not limited to glutathione-S-transferase (GST),
Horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta galactosidase, β-glucuronidase, fluorescent
Plain enzyme, green fluorescent protein (GFP), HcRed, DsRed, green fluorescin (CFP), yellow fluorescence protein (YFP), to include
The autofluorescence albumen of blue fluorescent protein (BFP).
C2c2 albumen can also merge a kind of protein or protein fragments, and the protein or protein fragments combine
DNA molecular combines other cellular elements comprising, but be not limited to, maltose-binding protein (MBP), S-tag, Lex A DNA
Binding structural domain (DBD) fusions, GAL4DNA binding structural domain fusions and herpes simplex virus (HSV) BP16 albumen
Fusions.In some embodiment of the invention, the fused protein is molecular labeling, be can be used to using the C2c2 albumen of label
Identify the position of target sequence.
Term " polypeptide ", " peptide " and " protein " is interchangeably used herein, refers to the amino acid with any length
Polymer.The polymer can be linear chain or branched chain, it may include the amino acid of modification, and it can be by
Non-amino acid is interrupted.These terms also cover the amino acid polymer being modified；These modification such as disulfide bond formations, sugar
Base, esterification (lipidation), acetylation, phosphorylation or any other modification, such as knot with detection molecules labeling component
It closes.
In a preferred embodiment of this invention, second regulating element is T7 promoter.
In a preferred embodiment of this invention, described first or two regulating element further include enhancer, internal ribosome
Entry site (IRES) and other expression control elements (such as transcription stop signals, such as polyadenylation signal and poly U
Sequence).Such adjusting sequence is for example described in Gaston Godel (Goeddel), " gene expression technique: Enzymology method " (GENE
EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY) 185, academic press (Academic Press) is holy
Ground Asia brother (San Diego), in California (1990).Regulating element includes that one nucleotides sequence of guidance is listed in many classes
Those of constitutive expression in the host cell of type sequence and instruct the nucleotide sequence only in certain host cells table
Up to those of sequence (for example, organizing specific type adjust sequence).Tissue-specific promoter can be instructed mainly in the interested phase
Hope the expression in tissue, the tissue such as muscle, neuron, bone, skin, blood, specific organ (such as liver, pancreas
Gland) or special cell type (such as lymphocyte).Regulating element can be in a manner of Temporal dependency (such as with the cell cycle
Dependence or stage of development dependence mode) guidance expression, which may or may not be that tissue or cell type are special
Anisotropic.
Fourth aspect present invention provides a kind of method for modifying sequence relevant to target nucleotide described in first aspect,
The method includes deliver the composition and the target nucleotide made comprising composition 1) and 2) comprising 1) and 2) and it is described with
The relevant sequence of target nucleotide is close:
1) C2c2 effect protein；
2) one or more nucleic acid, wherein one or more nucleic acid include it is at least one as described in relation to the first aspect
GRNA sequence；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 is compound
When object is in conjunction with target nucleotide described in first aspect, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide is modified.
In a preferred embodiment of this invention, the modification is to introduce fracture, cutting or label.
In one embodiment of fourth aspect present invention, the target nucleotide includes target nucleotide (target described in first aspect
) and/or reporter rna RNA.
In an embodiment of the present invention, component I further comprises be operably connected to first regulating element two
The coded sequence of a or more coding gRNA, when expression, each of two or more gRNA guidance
CRISPR-C2c2 compound specifically bound from different target nucleotide sequences (association reaction can occur in host cell,
In in-vitro transcription/translation system or other skilled in the art according to specific experiments demand configuration reaction solution).
In embodiments of the present invention, term " makes comprising composition and the target nucleotide and described and report 1) and 2)
It is close to accuse RNA " refer to component is delivered in vitro (in vitro) or in vivo (in vivo) environment, ex vivo environment such as ability
The field technique personnel reaction solution that demand configures according to specific experiments, vivo environment are such as intracellular；The term " close "
Refer in vitro (in vitro) or in vivo (in vivo) environment, each component can be with the target nucleotide and described and report
The sequence contact of RNA is accused, and the reaction that those skilled in the art are anticipated that occurs under certain conditions.
In embodiments of the present invention, the present invention provides following methods, including deliver one or more to host cell
Polynucleotides, one or more carriers, one or more transcripts, and/or the albumen of one or more transcriptions.In some sides
Face, invention further provides the cell generated by such method and including such cell or by such cell
The organism (such as animal, plant or fungi) of generation.
In embodiments of the present invention, the CRISPR-C2c2 compound combined with gRNA is delivered to cell.It can be used
Nucleic acid is introduced into mammalian cell or target tissue by the gene transfer method of conventional virus and non-viral base.
Cell of such method into culture or in host organism can be used and give coding CRISPR-C2c2 system
Component nucleic acid.Non-virus carrier delivery system includes DNA plasmid, RNA (such as transcript of carrier described herein), naked
Nucleic acid and the nucleic acid compound with Delivery excipient (such as liposome).Viral vector delivery system includes DNA and RNA virus,
They have sequestered or integrated genome after being delivered to cell.About the summary of genes delivery system, referring to peace moral
(Anderson), " science " (Science) 256:808-813 (1992)；Receive Bell (Nabel) Fil Ge Na (Felgner),
TIBTECH 11:211-217 (1993)；Three paddy (Mitani) Ka Siji (Caskey), TIBTECH 11:162-166
(1993)；Di Long (Dillon), TIBTECH 11:167-175 (1993)；Miller (Miller), " nature " (Nature) 357:
455-460(1992)；Fan Bulangte (Van Brunt), " biotechnology " (Biotechnology) 6 (10): 1149-1154
(1988)；Wei Nie (Vigne), " restoring neurology and Neuscience " (Restorative Neurology and
Neurosciece) (1995) 8:35-36；Cray is write from memory, and (Kremer) & Perry Coudé is special (Perricaudet), and " British Medical is public
Report " 51 (1) (British Medical Bulletin): 31-44 (1995)；Clatter clatter (Haddada) et al. is breathed out, in " microorganism
Learn and immunology current topic " Dorr husband strangles in (Current Topics in Microbiologyand Immunology)
(Doerfler) and rich nurse (editor) (1995)；And remaining (Yu) et al., " gene therapy " (Gene Therapy) 1:13-26
(1994)。
The Nonviral delivery methods of nucleic acid include fat transfection, nuclear transfection, microinjection, particle gun, virion, lipid
Body, immunoliposome, polycation or lipid: nucleic acid conjugate, naked DNA, artificial virions and DNA reagent enhancement take the photograph
It takes.Fat transfection is described in such as U.S. Patent number 5,049,386,4,946,787 and 4,897,355 and lipofectin reagent is
Commercially available (for example, TransfectamTM and LipofectinTM).Effective Receptor recognition fat transfection suitable for polynucleotides
Cation and neutral lipid include Felgner (Fil Ge Na), WO 91/17424；Those of WO 91/16024.Delivering can
For cell (such as giving in vitro or in vitro) or target tissue (such as giving in vivo).
Lipid: the preparation of nucleic acid complexes (liposome including targeting, such as immunolipid complexes) is the skill of this field
(see, for example, Krystal (Crystal), " science " (Science) 270:404-410 (1995) known to art personnel；Cloth
Lai Ze (Blaese) et al., " cancer gene therapy " (Cancer Gene Ther.) 2:291-297 (1995)；Bell (Behr)
Et al., " bioconjugate chemistry " (Bioconjugate Chem.) 5:382-389 (1994)；Thunder rice (Remy) et al., " biology is altogether
Yoke chemistry " 5:647-654 (1994)；High (Gao) et al., " gene therapy " (Gene Therapy) 2:710-722 (1995)；Chinese mugwort
Ha Maide (Ahmad) et al., " cancer research " (Cancer Res.) 52:4817-4820 (1992)；U.S. Patent number 4,186,
183,4,217,344,4,235,871,4,261,975,4,485,054,4,501,728,4,774,085,4,837,028 with
And 4,946,787).
Fifth aspect present invention provides a kind of eukaryotic host cell, includes component I and/or component II:
The component I includes the first regulating element, and the coding being operably connected with first regulating element
The coded sequence of C2c2 albumen；The component II includes the second regulating element, and operationally with second regulating element
The coded sequence of the coding gRNA of connection, wherein the gRNA includes gRNA sequence described in first aspect；
Wherein, component I and II is located on identical or different carrier；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 is compound
When object is in conjunction with target nucleotide described in first aspect, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide is modified.
In a preferred embodiment of this invention, the modification is to introduce fracture, cutting or label.
In one embodiment of fifth aspect present invention, the target nucleotide includes target nucleotide (target described in first aspect
) and/or reporter rna RNA.
In an embodiment of the present invention, the eukaryotic host cell includes component I and component II.
In an embodiment of the present invention, component I further comprises be operably connected to first regulating element two
The coded sequence of a or more coding gRNA, when expression, each of two or more gRNA guidance
CRISPR-C2c2 compound is specifically bound in eukaryotic host cell from different target nucleotide sequences.
Sixth aspect present invention provides a kind of detection kit, gRNA sequence, the second party provided comprising first aspect
In terms of non-naturally occurring or engineering the composition of CRISPR-C2c2 system, third aspect offer that face provides, the 5th
One of eukaryotic host cell of offer is a variety of.
In an embodiment of the present invention, the kit further includes conventional matched reaction reagent and/or consersion unit.Example
Such as, kit can provide one or more reactions or storage buffer.It by the available form in specific measurement or can press
The form (such as by concentration or lyophilized form) for needing to add one or more other components before the use provides reagent.Buffering
Liquid can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer liquid, borate buffer solution, Tris
Buffer, MOPS buffer, HEPES buffer solution and combinations thereof.In some embodiments, which is alkaline.Some
In embodiment, which has the pH from about 7 to about 10.In some embodiments, which includes one or more few
Nucleotide, one or more nucleic acid include at least one gRNA, and the gRNA includes gRNA sequence as described in relation to the first aspect.
Each component in kit of the present invention can provide either individually or in combination, and can be provided in any
In suitable container, such as bottle, bottle, pipe or cardboard.
Seventh aspect present invention provides a kind of detection side of tumour related mutation gene based on CRISPR-C2c2 system
Method includes:
1) prepare or provide sample to be tested, wherein the sample to be tested includes DNA and/or RNA；
2) it provides comprising composition a), b) and c), component a) includes C2c2 effect protein；Component b) include it is a kind of or
Multiple nucleic acids, wherein one or more nucleic acid include at least one gRNA, and the gRNA includes described in first aspect
GRNA sequence；Component c) includes the reporter rna for being modified with numerator detection mark；
3) in reaction system, make to contact comprising composition a) b) c) with the sample to be tested, the C2c2 albumen and
GRNA is combined and is formed CRISPR-C2c2 compound, and the CRISPR-C2c2 compound is and right in conjunction with the target nucleotide
The reporter rna for being modified with numerator detection mark is sheared, and the numerator detection mark that can be detected is generated；
4) numerator detection mark is detected, testing result is obtained.
In an embodiment of the present invention, the target nucleotide is to be connected with the DNA fragmentation of T7 promoter through T7 polymerase turn
Record the RNA obtained.
Optionally, the DNA fragmentation is extracted or purifying obtains, and is modified with T7 promoter.Optionally, it is described extraction or
The DNA fragmentation of purifying is modified with T7 and opens by PCR amplification, NASBA isothermal duplication or recombinase polymerase RPA amplification processing
Mover.
In an embodiment of the present invention, the reaction system includes Cas13a detection architecture.It is embodied in the present invention one
In example, the Cas13a detection architecture includes: the LwCas13a of 45nM purifying, and 22.5nM gRNA, 125nM is in LwCas13a
The reporter rna chain (RNAse Alert v2, Thermo Scientific) of capable of emitting fluorescence, 2 μ L small source of mouse RNase when cutting
Inhibitor (New England Biolabs), the total source of people RNA of 100ng (being purified from HEK293FT culture medium), the target of different number
Nucleic acid and nuclease detection buffer (40mM Tris-HCl, 60mM NaCl, 6mM MgCl2, pH 7.3).
In an embodiment of the present invention, the reaction system includes that RPA-DNA amplification system, T7 polymerase transcribe DNA
For RNA reaction system and Cas13a detection architecture.In a specific embodiment of the invention, the reaction system (50 μ L system)
It include: 0.48 μM of forward primer, 0.48 μM of reverse primer, 1xRPA fluid infusion buffer, different amounts of DNA, 45nM LwCas13a
Recombinant protein, 22.5nM gRNA, 250ng total people RNA, 200nM RNA report sub (RNase alert v2), 4 μ L source of mouse
RNase inhibitor (New England Biolabs), 2mM ATP, 2mM GTP, 2mM UTP, 2mM CTP, 1 μ LT7 polymerase
Mixture (New England Biolabs), 5mMMgCl2With 14mM MgAc.
Eighth aspect present invention provides a kind of detection examination of tumour related mutation gene based on CRISPR-C2c2 system
Agent box, the gRNA sequence provided including first aspect, the CRISPR-C2c2 system of second aspect offer, the third aspect provide
One of eukaryotic host cell that non-naturally occurring or engineering composition, the 5th aspect provide is a variety of.
In an embodiment of the present invention, kit that the eighth aspect provides further include: PCR amplification, NASBA isothermal
Amplification or recombinase polymerase RPA, ring mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification
(HDA) and one of nickase amplified reaction (NEAR) or plurality of reagents.
In an embodiment of the present invention, the kit that the eighth aspect provides further includes T7 polymerase.
Ninth aspect present invention provides a kind of application of gRNA sequence as described in relation to the first aspect, comprising:
(i) compound is formed with C2c2, binding molecule labelling technique, such as fluorescent labelling techniques show and the gRNA sequence
The target RNA of specific binding is arranged in transport and/or positioning intracellular；
(ii) compound is formed with C2c2, captures specific transcriptional object, the specific transcriptional object and the gRNA sequence
Biotin ligase activity (is navigated to specific transcription by the direct drop-down of dC2c2 or using dC2c2 by specific binding
Object).
Specific embodiment
As described below is the preferred embodiment of the embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, without departing from the principles of the embodiments of the present invention, several improvements and modifications can also be made, these improvement
Also it is considered as the protection scope of the embodiment of the present invention with retouching.
Unless otherwise noted, agents useful for same and consumptive material are commercial goods in the embodiment of the present invention.
Unless otherwise indicated, practice of the invention uses immunology, biochemistry, chemistry, molecular biology, microorganism
, cell biology, genomics and recombinant DNA routine techniques, these are within the technical ability of this field.Referring to Pehanorm cloth Shandong
Gram (Sambrook), not Ritchie (Fritsch) and the Germania base of a fruit this (Maniatis), " molecular cloning: laboratory manual "
(MOLECULAR CLONING:A LABORATORY MANUAL), the 2nd editor (1989)；" Current Protocols test hand
Volume " (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (F.M. Austria Su Beier (F.M.Ausubel) et al. is compiled
Volume, (1987))；" Enzymology method " (METHODS IN ENZYMOLOGY) series (Academic Press Inc): " PCR2: practical side
Method " (PCR 2:A PRACTICAL APPROACH) (M.J. McPpherson (M.J.MacPherson), B.D. Hei Musi
(B.D.Hames) and Taylor G.R. (G.R.Taylor) edit (1995)), Ha Luo (Harlow) and draw in (Labe) edit
(1988) " antibody: laboratory manual " (ANTIBODIES, A LABORATORY MANUAL), and " animal cell culture "
(ANIMAL CELL CULTURE) (R.I. Fu Leixieni (R.I.Freshney) edits (1987)).
In the embodiment of the invention, the embodiment of the invention provides one kind to be used for target tumor related mutation gene
The gRNA of RNA.The detection method of the embodiment of the invention also provides a kind of tumour related mutation gene based on C2c2, detection examination
Agent box, and by April 13rd, 2017, Science magazine delivers an entitled " Nucleic acid detection with
One or more steps in experimental method disclosed in CRISPR-Cas13a " article (subsequent to be known as " document 1 ") are by quoting simultaneously
Enter present embodiment.The including but not limited to one or more steps of following steps:
One, Cas13a (i.e. C2c2) gene cloning and protein expression
Using the Cas13a albumen base for being originated from Leptotrichia wadei F0279 and Leptotrichia shahii
Cause, codon optimization, makes gene be more suitable for expressing in mammalian cells.Cas13a protein gene cloning after optimization
Entering pACYC184 skeleton, (skeleton includes by the intervening sequence of J23119 promoter driving expression, and the intervening sequence is that β-is interior
Amidase targeting or non-targeted spacer region).
The Cas13a protein gene cloning of codon optimization is to prokaryotic expression plasmid vector, the prokaryotic expression plasmid
The pET plasmid with 6-His histidine tag can be used in carrier, and protein purification is facilitated to express.It expresses bacterium and uses Rosetta2
(DE3)。
Institute of the embodiment of the present invention using plasmid includes:
PC004 plasmid map: (the i.e. band beta-lactamase scanning site https: //benchling.com/s/1PJ1cCwR
PACYC184)
PC009 plasmid map: https: //benchling.com/s/seq-ylkMuglYmiG4A3VhShZg
(pACYC184 plasmid of the LshCas13a gene insertion with beta-lactamase scanning site)
PC010 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a
(insertion of LshCas13a gene is without β-lactamase scanning site pACYC184 plasmid)
PC011 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a
(pACYC184 plasmid of the LwCas13a gene insertion with beta-lactamase scanning site)
PC012 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a
(pACYC184 plasmid of the LwCas13a gene insertion without beta-lactamase scanning site)
PC013 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a
(LwCas13a gene is inserted into the pACYC184 plasmid with Twin-Strep label)
After the conversion of Cas13a Protein reconstitution expression vector, it is pure to carry out protein expression, SDS-PAGE detection and gel column
Change, the Cas13a albumen after purification of acquisition puts -80 DEG C of preservations.
Two, the preparation gRNA of target RNA prepares target nucleotide:
It extracts
Target nucleotide passes through PCR amplification, recombinase polymeric enzymatic amplification (RPA), NASBA isothermal duplication or ring mediated isothermal
Amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HDA) and nickase amplified reaction (NEAR) mode expand
Increase target DNA.Glue separation and purifying (using MinElute gel extraction kit (Qiagen) kit), after purification
DsDNA with 30 DEG C of T7 polymerase be incubated overnight (use HiScribe T7 Quick High Yield RNA Synthesis
Kit (New England Biolabs) kit), then use MEGAclear Transcription Clean-up kit
(Thermo Fisher) kits RNA, to obtain target nucleus RNA.
NASBA isothermal duplication
At 4 DEG C, configuration amplification system is as follows:
Above-mentioned mixed system places 2min under the conditions of 65 DEG C；Then at 41 DEG C, 2 minutes；
It is added in above-mentioned mixed system 5ul enzymatic mixture (Life Sciences, NEC-1-24), it is always anti-to obtain 20 μ L
Answer system.It is reacted 2 hours under the conditions of 65 DEG C.
Recombinase polymeric enzymatic amplification RPA (Reeombinase Polymerase Amplification)
RPA primer is designed using NCBI Primer blast, amplified fragments size is 80-180nt, the denaturation temperature of primer
Degree can be 54-67 DEG C, Opt=60, and length 30-35nt, Opt=32, G/C content is 40-60% in primer, according to designing sequence
Column synthetic DNA primer.
It refers to respectivelyBasic andBasicRT (TwistDx) kit carries out RPA reaction, no
It is same, before template segments addition, the MgAc of 280mM, i.e. magnesium acetate is first added.It is reacted 2 hours at 37 DEG C.
Three, the preparation of gRNA
Prepare gRNA: referring to HiSctibeT7 Quick High Yield RNA Synthesis kit (New
England Biolabs) kit specification, the DNA fragmentation with T7 promoter, T7 primer, T7 polymerase are mixed, 37 DEG C incubate
It educates overnight；RNAXP clean beads (Beckman Coulter) kits are used again, obtain the gRNAs of purifying.
Four, tumour related mutation gene is detected
Tumour related mutation genetic test system includes: the LwCas13a of 45nM purifying, and 22.5nM gRNA, 125nM exist
The reporter rna chain (RNAse Alert v2, Thermo Scientific) of LwCas13a capable of emitting fluorescence when cutting, 2 μ l mouse
Source RNase inhibitor (New England Biolabs), target RNA and nuclease detection buffer (40mM Tris-HCl,
60mM NaCl, 6mM MgCl 2, pH 7.3).Reaction system is placed in fluorescence analyser (BioTek), at 37 DEG C (unless otherwise saying
It is bright) under react 1-3 hours, fluorescence Dynamic testing 5 minutes is primary.
Five, SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) one
Footwork detects tumour related mutation gene
Optionally, DNA cloning above-mentioned, DNA is transcribed into RNA and Cas13a detection architecture by T7 polymerase to match
It sets in the same system and reacts.Optionally, which, which configures, includes:
In 50 μ L systems:
0.48 μM of forward primer, 0.48 μM of reverse primer, 1x RPA fluid infusion buffer, different amounts of DNA, 45nM
LwCas13a recombinant protein, 22.5nM gRNA, 250ng total people RNA, 200nM RNA report sub (RNase alert v2), 4 μ l
Source of mouse RNase inhibitor (New England Biolabs), 2mM ATP, 2mM GTP, 2mM UTP, 2mM CTP, 1 μ l T7 are poly-
Synthase mixture (New England Biolabs), 5mM MgCl2With 14mM MgAc.Reaction system is placed in fluorescence analyser
(BioTek), it is reacted 1-3 hours under 37 DEG C (unless otherwise indicated), fluorescence Dynamic testing 5 minutes primary.
Six, SHERLOCK (Speeific High Sensitivity Enzymatic Reporter UnLOCKing) is cold
Dry and paper deposition is lyophilized
By glass fiber filter paper (Whatman, 1827-021) high pressure sterilization 90 minutes (Consolidated Stills and
Fungicide, MKII) and the closing in the BSA of 5% nuclease free (EMD Millipore, 126609-10GM), overnight.With seedless
After water (Life technologies, AM9932) the cleaning paper of sour enzyme is primary, by with 4%RNAsecure TM (Life
Technologies, AM7006) be incubated for (60 DEG C) removings ribalgilase 20 minutes, with the water of nuclease free flushing paper 3 times with
Remove the trace of RNAsecure.Paper is handled before use, first 20 minutes dry on hot plate at 80 DEG C (Cole-Parmer,
IKA C-Mag HS7).1.8 μ LCas13a reaction mixtures (as previously described) are placed on black, 384 orifice plate of clear bottom
On disk (2mm) in (Corning, 3544).For the freeze-drying of SHERLOCK is tested, the plate containing reaction mixture disk
It is rapidly frozen in liquid nitrogen, as previously mentioned, lyophilized overnight.RPA sample dilutes 1: 10 in the water of nuclease free, and will
The mixture of 1.8 μ L is loaded on paper disc and is incubated at 37 DEG C using orifice detector (plate reader, BioTek Neo)
It educates.
Seven, SHERLOCK fluorescence data is analyzed
In order to calculate the fluorescence data of removal background, facilitate the comparison between different condition, the initial fluorescence of sample is gone
It removes.Background fluorescence (no target nucleotide or without gRNA under conditions of) can be removed from sample, to obtain background correction fluorescence
Data.
It will be appreciated by persons skilled in the art that can be implemented using the alternative replacement present invention of this field routine
The conventional clone of Cas13a gene, the building of recombinant expression carrier, the expression of Cas13a albumen and purifying, target nucleus glycosides in example
The amplification of acid/target gene segment and etc. one of or it is a variety of, to obtain similar or equivalent effect.
It will be appreciated by persons skilled in the art that as disclosed in document 1: for different target nucleotides, gRNA with
And the sequence of Protospacer flanking site (PFS) is extremely important.PFS is target site existing specificity nearby
Motif is necessary to the strong ribonuclease activity of Cas13a.Although this motif is similar to PAM sequence, PAM is DNA target
To the second class crispr-cas system important sequence, but PFS and PAM is functionally different, because PFS is not involved in
Prevent self targeting of CRISPR locus in endogenous system.Importance of the PFS to Cas13a: such as gRNA target complex
It is formed and needs following more researchs with the influence in cleavage activity.
GRNA provided in an embodiment of the present invention for target tumor related mutation gene RNA, the tumour phase based on C2c2
The tumour related mutation gene of the detection method, detection kit detection of closing mutated gene includes but is not limited to table 1, shown in table 2
Tumour related mutation gene.
In the embodiment of the present invention, the capital and small letter of each base does not have particular meaning in table 1,2,3.Those skilled in the art can be with
Understand, the size of each base can be become small letter or small letter from capitalization in table of embodiment of the present invention 1-3 becomes size, meaning
It is constant.
In the embodiment of the present invention, the gene of tumour related mutation shown in the following table 1 is additionally provided, the mutagenic origin in table 1 is in (1)
50 base Substitutions or (2) national tumour germline mutation high-flux sequence detection data before MSK-IMPACT data set；Specifically
Ground, the mutational site of each tumour related mutation gene covering and genebank accession number are as shown in table 1 in table 1:
The mutational site of each tumour related mutation gene of table 1. covering and genebank accession number
In the embodiment of the present invention, the mutational site of each tumour related mutation gene covering and genebank accession number such as table
Shown in 2:
The corresponding mutational site of each tumour related mutation gene of table 2. and genebank accession number
In the embodiment of the present invention, for each tumour related mutation gene shown in Tables 1 and 2, provide special
Property identify one or more gRNA sequence of the tumour related mutation gene, as shown in table 3:
Claims (10)
1. a kind of gRNA sequence of specific recognition target nucleotide, which is characterized in that the target nucleotide is prominent for tumour correlation
Become the corresponding RNA sequence of gene, the corresponding DNA sequences encoding of the gRNA includes SEQ ID NO.1-SEQ ID in table 3
One or more in nucleotide sequence shown in NO.306.
2. a kind of CRISPR-C2c2 system characterized by comprising
1) C2c2 effect protein；
2) one or more nucleic acid, wherein one or more nucleic acid include at least one gRNA as described in claim 1
Sequence；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 compound with
When target nucleotide described in claim 1 combines, the CRISPR-C2c2 compound is to the target nucleotide and/or and target nucleus
The relevant sequence of thuja acid is modified.
3. a kind of non-naturally occurring or engineering composition, which is characterized in that the composition includes one or more carries
Body, the one or more carrier include component I and component II:
The component I includes the first regulating element, and the coding C2c2 egg being operably connected with first regulating element
White coded sequence；The component II includes the second regulating element, and be operably connected with second regulating element
Encode the coded sequence of gRNA, wherein the gRNA includes gRNA sequence as described in claim 1；
Wherein, component I and II is located on identical or different carrier；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 compound with
When target nucleotide described in claim 1 combines, the CRISPR-C2c2 compound is to the target nucleotide and/or and target nucleus
The relevant sequence of thuja acid is modified.
4. a kind of method for modifying sequence relevant to target nucleotide described in claim 1, which is characterized in that the method packet
Include delivering and include composition 1) and 2), make comprising 1) and 2) composition and the target nucleotide and described and target nucleotide phase
The sequence of pass is close:
1) C2c2 effect protein；
2) one or more nucleic acid, wherein one or more nucleic acid include at least one gRNA as described in claim 1
Sequence；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 compound with
When target nucleotide described in claim 1 combines, the CRISPR-C2c2 compound is to the target nucleotide and/or and target nucleus
The relevant sequence of thuja acid is modified.
5. a kind of eukaryotic host cell, which is characterized in that include component I and/or component II:
The component I includes the first regulating element, and the coding C2c2 egg being operably connected with first regulating element
White coded sequence；The component II includes the second regulating element, and be operably connected with second regulating element
Encode the coded sequence of gRNA, wherein the gRNA includes gRNA sequence described in claim 1；
Wherein, component I and II is located on identical or different carrier；
The C2c2 albumen forms CRISPR-C2c2 compound in conjunction with gRNA, and when the CRISPR-C2c2 compound with
When target nucleotide described in claim 1 combines, the CRISPR-C2c2 compound is to the target nucleotide and/or and target nucleus
The relevant sequence of thuja acid is modified.
6. such as any one of claim 2-5, which is characterized in that the modification is to introduce fracture, cutting or label.
7. a kind of detection kit, which is characterized in that gRNA sequence, the claim 2 provided including claim 1 provides
CRISPR-C2c2 system, non-naturally occurring or engineering the composition of claim 3 offer, claim 5 provide true
One of core host cell is a variety of.
8. a kind of detection method of the tumour related mutation gene based on CRISPR-C2c2 system, characterized by comprising:
1) prepare or provide sample to be tested, wherein the sample to be tested includes DNA and/or RNA；
2) it provides comprising composition a), b) and c), component a) includes C2c2 effect protein；Component b) includes one or more
Nucleic acid, wherein one or more nucleic acid include at least one gRNA, and the gRNA includes gRNA described in claim 1
Sequence；Component c) includes the reporter rna for being modified with numerator detection mark；
3) in reaction system, make to contact comprising composition a) b) c) with the sample to be tested, the C2c2 albumen and gRNA are tied
Conjunction forms CRISPR-C2c2 compound, and the CRISPR-C2c2 compound is repaired in conjunction with the target nucleotide, and to described
The reporter rna for being decorated with numerator detection mark is sheared, and the numerator detection mark that can be detected is generated；
4) numerator detection mark is detected, testing result is obtained.
9. a kind of detection kit of the tumour related mutation gene based on CRISPR-C2c2 system, which is characterized in that including power
The non-natural of CRISPR-C2c2 system, claim 3 offer that benefit requires the gRNA sequence of 1 offer, claim 2 to provide is deposited
Or one of eukaryotic host cell that the composition of engineering, claim 5 provide or a variety of；It further include T7 polymerization
Enzyme.
10. a kind of application of gRNA sequence as described in claim 1 characterized by comprising
(i) compound is formed with C2c2, binding molecule labelling technique shows the target in conjunction with the gRNA sequence-specific
RNA is in transport and/or positioning intracellular；
(ii) compound is formed with C2c2, captures specific transcriptional object, the specific transcriptional object and the gRNA sequence specific
Property combine.
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