CN109402115A - Target Rett mutated gene RNA gRNA and Rett mutated gene detection method, detection kit - Google Patents
Target Rett mutated gene RNA gRNA and Rett mutated gene detection method, detection kit Download PDFInfo
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Abstract
The present invention provides a kind of for targeting the gRNA of Rett syndrome related mutation gene RNA, and the present invention also provides a kind of people's Rett syndrome 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 Rett syndrome 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 Rett syndrome 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 related for selectively targeted Rett syndrome
The gRNA of mutated gene RNA, and a kind of Rett based on short palindrome repetitive sequence (CRISPR) system of regular intervals cluster are comprehensive
Disease related mutation gene tester, detection kit；In particular to a kind of to be used for selectively targeted Rett syndrome resistance base
The gRNA of cause and/or specific gene RNA, and one kind is based on short palindrome repetitive sequence (CRISPR) system of regular intervals cluster
Rett syndrome resistant gene and/or specific gene detection method, detection kit.
Background technique
CRISPR/Cas is the strong tools for carrying out gene editing, can be to the accurate edits that gene is pinpointed.
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: CRISPR-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.
Rett's syndrome, Rett Syndrome, it is estimated that, China annual about 1,000 suffers from Rett syndrome
Girl baby's birth.The girl of illness seeming that development is normal during big by 6 months from birth, and symptom is big at 6 to 18 months
When start to show, it is mechanical including the communication capability grasped before losing and purposive hand function, the repetition for hand occur
It acts (such as write, pat, stinging hand), as other symptom of auxiliary diagnosis, that there are also respiratory rhythms is abnormal, grinds one's teeth in sleep, vertebra side
Curved, epilepsy and growth retardation.The clinical symptoms of Rett syndrome are changeable, partly because different types of change on MECP gene
It is different to cause.Most of (80%) adult patients have the symptom of epilepsy, and with age, the spasm activity of some women is changed
It is kind, but some situations are difficult treatment, need drug control mostly.
Applicants have found that not there is the Rett syndrome related mutation gene based on CRISPR-C2c2 system also at present
The relevant report of detection method.GRNA as designing, preparing accurate, selectively targeted target gene is CRISPR-Cas9
The gRNA of the key technology of gene knockout, efficient, selectively targeted target Rett syndrome related mutation gene is also
CRISPR-C2c2 identifies the key of target gene, and further such that the Rett syndrome correlation based on CRISPR-C2c2 system is prominent
The editor of change gene, modification, detection are possibly realized.
Therefore, intend applying for a kind of Rett syndrome related mutation gene tester based on CRISPR-C2c2 system, inspection
Test agent box and gRNA for selectively targeted Rett syndrome 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 one kind to be used for selectively targeted Rett syndrome related mutation gene
The gRNA of RNA and the Rett syndrome 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 of specific recognition target nucleotide, and the target nucleotide is
The corresponding RNA sequence of Rett syndrome related mutation gene, the corresponding DNA sequences encoding of the gRNA includes SEQ ID in table 1
One or more in nucleotide sequence shown in NO.1-SEQ ID NO.28.
In one embodiment of the invention, the Rett syndrome related mutation gene be Rett syndrome resistant gene and/or
Rett syndrome specific gene.
In one embodiment of the invention, the Rett syndrome related mutation gene includes in table 1 shown in gene number 1-14
The one or more of gene.
In one embodiment of the invention, " the corresponding RNA sequence of Rett syndrome related mutation gene " is comprehensive for Rett
The corresponding transcript of disease 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 related for each Rett syndrome shown in gene number 1-14 in table 1
Mutated gene devises a plurality of gRNA, every gRNA can independently, the corresponding Rett syndrome phase of the specific recognition row
Close mutated gene.
For example, the Rett syndrome related mutation gene is MECP2, the corresponding DNA sequences encoding of gRNA includes in table 1
One or more in nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.2.The Rett syndrome related mutation base
Because the corresponding DNA sequences encoding of MECP2, gRNA includes nucleotide sequence shown in SEQ ID NO3-SEQ ID NO.4 in table 1
In one or more.And so on, it will be appreciated by persons skilled in the art that every shown in gene coding 1-14 in table 1
One Rett syndrome related mutation gene respectively corresponds one or more gRNA in table 1.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, and b is g or c or t, and d is a or g or t, and v is a or g or c, and 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 such as SEQ ID NO.1- in table 1
SEQ ID NO.28 shows one or more in nucleotide sequence.
Common gRNA frame sequence includes but is not limited to: 5 '-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAA
AC。
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 answered with PFS segment (similar PAM) by Cas13a-gRNA
GRNA specific recognition in zoarium.
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
It is located on identical or different carrier with II；
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 promoter),
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 includes
But it is 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: 1,2 CRISPR- of Discovery and Functional Characterization of Diverse Class
Cas Systems, Nature, 2016 Oct 13；2,C2c2 is a single-component programmable RNA-
Guided RNA-targeting CRISPR effector, 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 for 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 includes, but are not limited to glutathione-S-transferase (GST), peppery
Root peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta galactosidase, β-glucuronidase, luciferin
Enzyme, green fluorescent protein (GFP), HcRed, DsRed, green fluorescin (CFP), yellow fluorescence protein (YFP) including blue
The autofluorescence albumen of fluorescin (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 such as
GRNA sequence described in first aspect；
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)8:35-36(1995)；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
It is mentioned 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 confession 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 Rett syndrome related mutation gene based on CRISPR-C2c2 system
Detection 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 MgCl 2, 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, 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 LT7
Polymerase mixture (New England Biolabs), 5mMMgCl2With 14mM MgAc.
Eighth aspect present invention provides a kind of Rett syndrome related mutation gene based on CRISPR-C2c2 system
Detection kit, CRISPR-C2c2 system, the third aspect that the gRNA sequence provided including first aspect, second aspect provide
One of eukaryotic host cell of non-naturally occurring or engineering the composition, the 5th aspect offer that 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 transcript by the direct drop-down of dC2c2 or using dC2c2) by specific binding.
Detailed description of the invention
Fig. 1 is the testing result of the Rett syndrome related mutation gene provided in an embodiment of the present invention based on C2c2.
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 (Lane) 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, it is prominent for targeting Rett syndrome correlation that the embodiment of the invention provides one kind
Become the gRNA of gene RNA.The inspection of the embodiment of the invention also provides a kind of Rett syndrome related mutation gene based on C2c2
Survey method, detection kit, and by April 13rd, 2017, Science magazine delivers an entitled " Nucleic acid
One or more in experimental method disclosed in detection with CRISPR-Cas13a " article (subsequent to be known as " document 1 ")
A step is incorporated by reference into 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)。
The used plasmid of the embodiment of the present invention includes: pC004 plasmid map: https: //benchling.com/s/
LPJ1cCwR (pACYC184 i.e. with beta-lactamase scanning site)；PC009 plasmid map: https: //
(LshCas13a gene insertion band beta-lactamase scans position to benchling.com/s/seq-ylkMuglYmiG4A3VhShZg
The pACYC184 plasmid of point)；PC010 plasmid map: https: //benchling.com/s/seq-
2WApFr3zni1GOACyQY8a (pACYC184 plasmid of the LshCas13a gene insertion without beta-lactamase scanning site)；
PC011 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a (LwCas13a gene insert
Enter the pACYC184 plasmid with beta-lactamase scanning site)；PC012 plasmid map: https: //benchling.com/s/
Seq-2WApFr3zni1GOACyQY8a (pACYC184 matter of the LwCas13a gene insertion without beta-lactamase scanning site
Grain)；PC013 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a (LwCas13a base
Because being inserted into the pACYC184 plasmid with Twin-Strep label)；After the conversion of Cas13a Protein reconstitution expression vector, albumen is carried out
Expression, SDS-PAGE detection and gel column purification, the Cas13a albumen after purification of acquisition put -80 DEG C of preservations.
Two, the preparation gRNA of target RNA prepares target nucleotide:
It extracts target nucleotide and passes through PCR amplification, recombinase polymeric enzymatic amplification (RPA), NASBA isothermal duplication or ring mediate
Isothermal duplication (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HDA) and nickase amplified reaction (NEAR) side
Formula expands target DNA.Glue separation and purifying (using MinElute gel extraction kit (Qiagen) kit), it is pure
DsDNA after change is incubated overnight with 30 DEG C of T7 polymerase (uses 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 (Recombinase 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 HiScribeT7 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, Rett syndrome related mutation gene is detected
Rett syndrome related mutation genetic test system includes: LwCas13a, the 22.5nM gRNA of 45nM purifying,
Reporter rna chain (RNAse the Alert v2, Thermo of 125nM capable of emitting fluorescence when LwCas13a is cut
Scientific), the small source of mouse RNase inhibitor of 2 μ l (New England Biolabs), target RNA and nuclease detection buffering
Liquid (40mM Tris-HCl, 60mM NaCl, 6mM MgCl 2, pH 7.3).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.
Five, SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) one
Footwork detects Rett syndrome 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 (Specific 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 nothing
After water (Life technologies, AM9932) the cleaning paper of nuclease 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, elder generation dry 20 minutes (Cole-Parmer, IKA on hot plate at 80 DEG C
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.
It is comprehensive provided by the present invention for gRNA, the Rett based on C2c2 of targeting Rett syndrome related mutation gene RNA
Close the detection method of disease related mutation gene, the Rett syndrome related mutation gene that detection kit detects includes but is not limited to
Rett syndrome related mutation gene shown in table 1.
In the embodiment of the present invention, the capital and small letter of each base does not have particular meaning in table 1.It will be appreciated by those skilled in the art that
, the size of each base can be become small letter or small letter from capitalization in table of the embodiment of the present invention 1 becomes size, and meaning is constant.
In the embodiment of the present invention, for each Rett syndrome related mutation gene, provide described in specific recognition
One or more gRNA sequence of Rett syndrome related mutation gene, as shown in table 1:
Effect example
In the embodiment of the invention, it is prominent for targeting Rett syndrome correlation that the embodiment of the invention provides one kind
Become detection method, the detection kit of the gRNA of gene RNA, one kind and the Rett syndrome related mutation gene based on C2c2,
The including but not limited to one or more steps of following steps:
The present embodiment is only used as a kind of specific implementation of technical solution of the present invention, does not limit protection of the invention specifically
Range.
One, Cas13a (i.e. C2c2) gene cloning and protein expression
Cas13a (i.e. C2c2) gene cloning and protein expression, Activity determination (efficiency assay) reference
" Nucleic acid detection with CRISPR-Cas13a, Jonathan S Gootenberg, Science,
2017.4.13 " experimental method and step disclosed in (document 1).
If the conventional reagent that the present invention uses is commercial goods without specified otherwise.
The present embodiments relate to relevant primer explanation:
With gRNA relevant primer sequence:
T7Lwa DRgRNA FP:
TAATACgACTCACTATAggggggATTTAgACTACCCCAAAAACgAAggggACTAAAAC
CrRNA/gRNA primer:
The reverse complementary sequence-of gRNA sequence shown in SEQ ID NO.1-28 in 5 '-tables 1
GTTTTAGTCCCCTTCGTTTTTGGGGTAGTCT-3’。
Primer sequence relevant with target sequence:
T7FP:TAATACgACTCACTATAggg
Wt target primer sequence (gene order of wt expression normal person):
GRNA sequence (the SEQ ID NO.1-28 in table 1 shown in SEQ ID NO.1-28 in the corresponding table 1 of 5 '-TCGAG-
Shown gRNA sequence is respectively its saltant type, and " correspondence " refers to the corresponding wild type of saltant type, that is, corresponds to table 1 in genebank
Or the gene order of normal person disclosed in ncib)-
ATTTAGCCCTATAGTGAGTCGTATTA-3’。
Mut target primer sequence:
GRNA sequence-shown in SEQ ID NO.1-28 in 5 '-TCGAG- tables 1
ATTTAGCCCTATAGTGAGTCGTATTA-3’。
Using the Cas13a protein gene for being originated from Leptotrichia wadei F0279, codon optimization makes base
Because being more suitable for expressing in mammalian cells.Cas13a protein gene cloning after optimization enters pACYC184 skeleton (skeleton
Including the intervening sequence by the driving expression of J23119 promoter, the intervening sequence is beta-lactamase targeting or non-targeted interval
Area).
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)。
The used plasmid of the embodiment of the present invention includes: pC004 plasmid map: https: //benchling.com/s/
LPJ1cCwR (pACYC184 i.e. with beta-lactamase scanning site)；PC009 plasmid map: https: //
(LshCas13a gene insertion band beta-lactamase scans position to benchling.com/s/seq-ylkMuglYmiG4A3VhShZg
The pACYC184 plasmid of point)；PC010 plasmid map: https: //benchling.com/s/seq-
2WApFr3zni1GOACyQY8a (pACYC184 plasmid of the LshCas13a gene insertion without beta-lactamase scanning site)；
PC011 plasmid map: https: //benchling.com/s/seq-2WApFr3zni1GOACyQY8a (LwCas13a gene
It is inserted into the pACYC184 plasmid with beta-lactamase scanning site)；PC012 plasmid map: https: //benchling.com/
S/seq-2WApFr3zni1GOACyQY8a (pACYC184 matter of the LwCas13a gene insertion without beta-lactamase scanning site
Grain)；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, carry out
Protein expression, SDS-PAGE detection and gel column purification, the Cas13a albumen after purification of acquisition put -80 DEG C of preservations.
Two, template pre-treatment in site to be detected
10 μM of T7 primers, 10 μM of target primers, the 2x KOD FX μ L KOD FX of buffer, 200 μM of dNTP, 0.1 (KOD
FX, KFX-101, TOYOBO), add water to 10 μ L.With PCR program: 94 DEG C of 10s, 60 DEG C of 10s, 68 DEG C of 20s expand 6 circulations.Instead
50 μ L should be added water to after the completion, -20 DEG C save backup.
It is respectively synthesized target primer sequence, each target primer sequence separately includes shown in the SEQ ID NO.1-28 in table 1
GRNA sequence.
Three, prepared by crRNA
10 μM of T7 primers, 10 μM of crRNA primers, the 2x KOD FX μ L KOD of buffer, 200 μM of dNTP, 0.5 FX
(KOD FX, KFX-101, TOYOBO), adds water to 50 μ L.With PCR program: 94 DEG C of 10s, 60 DEG C of 10s, 68 DEG C of 20s expand 6
Circulation.With Qiaquick PCR Purification kit (Qiaquick PCR Purification, 28104,
Qiagen product purification is carried out) with 15 μ L TE elution.It is transcribed in vitro: 2 μ g PCR products, 10 μ L 5x transcription
Buffer, ATP, GTP, UTP, CTP (NTP Set, 100mM Solution, R0481, Thermo) each 1 μ L, 1.5 μ L T7 RNA
Polymerase (T7 RNA Polymerase (20U/ μ L), EP0111, Thermo), adds water to 50 μ L.37 DEG C are reacted 16 hours.Instead
37 DEG C of 2 μ L Turbo DNase (TURBO DNase (2U/ μ L), AM2239, Thermo) are added after answering to be incubated for 2 hours.
Above-mentioned reaction product provides scheme with RNeasy Mini Kit (Rneasy Mini Kit, 76106, Qiagen) with kit
It is purified, and with 20 μ L RNase free water elution crRNA, -20 DEG C are saved backup.
It is respectively synthesized crRNA primer sequence, each crRNA primer sequence separately includes and the SEQ ID NO.1-28 in table 1
Sequence (special instruction, the gRNA sequence shown in the SEQ ID NO.1-28 in table 1 of the present invention of shown gRNA sequence reverse complemental
It is classified as the coded sequence of gRNA, needs that gRNA could be obtained by transcription)
Four, detection architecture (50 μ L)
1 μ L sample to be tested, 45nM LwCas13a, 22.5nM crRNA, 25ng mankind's total serum IgE, 125nM
Substrate reporter (RNaseAlert Lab Test Kit v2,4479768, Thermo), buffer buffer
(20mM HEPES, 60mM NaCl, 6mM MgCl2, pH 6.8), 1 μ L RNase inhibitor (RNasin Ribonuclease
Inhibitors, N2515, Promega), 1mM ATP, 1mM GTP, 1mM UTP, 1mM CTP (NTP Set, 100mM
Solution, R0481, Thermo), 1.5 μ L T7 RNA polymerases (T7 RNA Polymerase (20U/ μ L), EP0111,
Thermo)。
Five, read plate
It is recorded with VICTOR X5 plate reading machine, 490/ launch wavelength 520 of excitation wavelength is read, the reading duration from hole top
Every hole 1 second, plate support temperature are set as 37 DEG C, and the numerical value of reading in every 5 minutes is continuous to record 2 hours.
The present embodiment repeats said effect embodiment for every gRNA sequence shown in the SEQ ID NO.21-30 in table 2
Step 2 is repeated 3 times and is averaged, after fluorescence intensity removal background as shown in Figure 1 to five.In Fig. 1, abscissa is in table 1
The SEQ ID NO. of SEQ ID NO.1-28 is numbered；Ordinate is the fluorescence intensity for removing background.Wt is corresponding for target sequence to be measured
Normal person gene, Mut be the corresponding saltant type of target sequence.
As shown in Figure 1, different gRNA is different to the gene of testing gene normal person and the discrimination of saltant type: having
The specificity of a little gRNA is very strong, more can specifically identify SEQ ID NO.18 in saltant type, such as attached drawing 1,26,
Fluorescent differences reach 2 times or more；The specificity of some gRNA is stronger, can distinguish saltant type and the gene of normal person well,
For example SEQ ID NO.6,12,16,22 in attached drawing 1, fluorescent differences reach 1.5-2 times or so；The specificity of some gRNA is relatively
Difference has SEQ ID NO.2,8,24,28 in a degree of identification, such as attached drawing 1 to the gene of saltant type and normal person,
Fluorescent differences reach 1.2-1.5 times or so；Remaining difference between 1-1.2 times, such as SEQ ID NO.4 in attached drawing 1,10,
14、20。
In practical applications, fluorescent differences can satisfy the detection of basic difference degree, specifically to examine at 1.2 times or more
The purpose and requirement of survey.
It is attached: for further illustrate beneficial effects of the present invention, the present invention win the reading of step 5 as a result, statistics it is as follows,
1 corresponding data of attached drawing includes:
| mean | PC | >chrX:153296192 | >chrX:153296495 | >chrX:153296761 | >chrX:153296777 |
| Mutant | 0 | 37565.75 | 12834 | 36825.25 | 15492.25 |
| WT | 67447 | 24688.25 | 11819 | 22144.5 | 10021.75 |
| mean | >chrX:153296777 | >chrX:153296903 | >chrX:153296903 | >chrX:153296903 | >chrX:153296904 |
| Mutant | 17430.25 | 48986.75 | 46252.75 | 50515 | 39465 |
| WT | 15943.25 | 28839.5 | 42948.25 | 32532.75 | 17589.5 |
| mean | >chrX:153296904 | >chrX:153297761 | >chrX:153297889 | >chrX:153297889 | >chrX:153297971 |
| Mutant | 14993.5 | 29783.5 | 29211.75 | 30103.5 | 14855.25 |
| WT | 13140.25 | 16547.5 | 22295 | 14348.5 | 11454.5 |
Sequence table
<110>Guangzhou Hua Chang investment consultation Co., Ltd
<120>target Rett mutated gene RNA gRNA and Rett mutated gene detection method, detection kit
<130> 2018
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ctahgggggt gaggaggcgc tgctgctg 28
<210> 2
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ctaghggggt gaggaggcgc tgctgctg 28
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cctabgggtc ggcctcagct tttcgctt 28
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cctaahggtc ggcctcagct tttcgctt 28
<210> 5
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tachtggttt ctgctctcgc cgggaggg 28
<210> 6
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tacgvggttt ctgctctcgc cgggaggg 28
<210> 7
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ctcadcggga ggggctccct ctcccagt 28
<210> 8
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ctcacdggga ggggctccct ctcccagt 28
<210> 9
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ctctdcggga ggggctccct ctcccagt 28
<210> 10
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctctcdggga ggggctccct ctcccagt 28
<210> 11
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
acahtgggga caaacagaaa gacacaag 28
<210> 12
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
acagvgggga caaacagaaa gacacaag 28
<210> 13
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
acchtgggga caaacagaaa gacacaag 28
<210> 14
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
accgvgggga caaacagaaa gacacaag 28
<210> 15
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
acghtgggga caaacagaaa gacacaag 28
<210> 16
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acggvgggga caaacagaaa gacacaag 28
<210> 17
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ctavggggac aaacagaaag acacaagg 28
<210> 18
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ctathgggac aaacagaaag acacaagg 28
<210> 19
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ctcvggggac aaacagaaag acacaagg 28
<210> 20
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ctcthgggac aaacagaaag acacaagg 28
<210> 21
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
tcadcggtca cggatgatgg agcgccgc 28
<210> 22
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
tcacdggtca cggatgatgg agcgccgc 28
<210> 23
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ctcbtggctg cacgggctca tgcttgcc 28
<210> 24
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
ctcavggctg cacgggctca tgcttgcc 28
<210> 25
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
cttbtggctg cacgggctca tgcttgcc 28
<210> 26
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
cttavggctg cacgggctca tgcttgcc 28
<210> 27
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
cctahaggcc ctggaggtcc tggtcttc 28
<210> 28
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cctagbggcc ctggaggtcc tggtcttc 28
Claims (10)
1. a kind of gRNA sequence of specific recognition target nucleotide, which is characterized in that the target nucleotide is Rett syndrome
The corresponding RNA sequence of related mutation gene, the corresponding DNA sequences encoding of the gRNA includes SEQ ID NO.1-SEQ in table 1
One or more in nucleotide sequence shown in ID NO.28.
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 works as the CRISPR-C2c2 compound
When in conjunction with target nucleotide described in claim 1, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide 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 works as the CRISPR-C2c2 compound
When in conjunction with target nucleotide described in claim 1, the CRISPR-C2c2 compound is to the target nucleotide and/or and target
The relevant sequence of nucleotide 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 Rett syndrome related mutation gene based on CRISPR-C2c2 system, which is characterized in that packet
Contain:
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 Rett syndrome related mutation gene based on CRISPR-C2c2 system, which is characterized in that
CRISPR-C2c2 system, the claim 3 that gRNA sequence, the claim 2 provided including claim 1 provides provide non-
One of eukaryotic host cell that naturally occurring or engineering composition, claim 5 provide is a variety of；It further include T7
Polymerase.
10. a kind of application of gRNA sequence as described in claim 1 characterized by comprising
(i) gRNA and C2c2 forms compound, and binding molecule labelling technique shows in conjunction with the gRNA sequence-specific
Target RNA in transport and/or positioning intracellular；
(ii) gRNA and C2c2 forms compound, captures specific transcriptional object, the specific transcriptional object and the gRNA
Sequence-specific combines.
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