CN109666662A - Application of the novel ScCas12a in terms of detection of nucleic acids - Google Patents
Application of the novel ScCas12a in terms of detection of nucleic acids Download PDFInfo
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Abstract
The invention discloses application of the novel ScCas12a albumen in terms of detection of nucleic acids.The research of the invention finds that a kind of novel ScCas12a albumen with DNA cleavage activity, novel C RISPR/ScCas12a system be can be used as applied to detection of nucleic acids, gene editing, gene modification, provide a kind of new essential tool selection for gene editing, modification and Molecular Detection based on Cas12a.Provide a kind of novel nucleic acids detection system and kit including ScCas12a albumen and gRNA simultaneously, can in 25 DEG C -37 DEG C of realization at room temperature highly sensitive, high-precision Molecular Detection, detect specific good, high sensitivity, it is low in cost、It is easy to operate、Fast, have a wide range of application, had a good application prospect in terms of detection of nucleic acids.
Description
Technical field
The invention belongs to technical field of molecular biology.More particularly, to a kind of novel ScCas12a in detection of nucleic acids
The application of aspect.
Background technique
To realize that highly sensitive, high-precision of pathogen etc. detect, developing low-cost, accurate, efficient, quick diagnostic method
It is extremely important.In terms of pathogen detection, the classical phenotypic assay measurement neurological susceptibility or resistance, ELISA based on culture is used
Etc. be clinical pathogens detection used in conventional method.But since pathogen can not fully be cultivated in vitro,
The detection sensitivities such as GAP-associated protein GAP, small molecule are limited, and traditional detection method is time-consuming and accuracy is not high.And based on molecule
The speed and accuracy of detection can be improved in diagnostic method, this in hospital and community environment infection control, prevent, treat very
It is significant.
Technology currently used for such as pathogen detection etc. has: (1) it is separately cultured technology: pathogen isolation culture, especially
Pathogenic microorganism and viral are separately cultured, are the pathogen goldstandard identification technologies of early stage.This method exists to asking
Topic mainly has: it is separately cultured that time-consuming, generally requires several days time, cannot achieve and obtain testing result in the short time rapidly, and
Testing laboratory's hardware and experiment operator condition must be highly relied on, and is not suitable for there are not maturation culture means at present
The detection of pathogenic microorganism and virus.(2) immunology detection: with the immune response based on Ag-Ab, cause of disease correlation egg is identified
It is white, pathogen is detected from protein level.That there are detection sensitivities is lower for this method, and specificity by environment etc. influence compared with
Greatly, the window phase of detection is longer, is unable to satisfy diagnosis and treatment demand, is only applicable to primary dcreening operation and cannot function as the foundation made a definite diagnosis in time, no
The problems such as capable of identifying the different subtype of same class cause of disease.(3) polymerase chain reaction (PCR): including regular-PCR, allele
Specific PCR, real-time fluorescence quantitative PCR, PCR-Sanger sequencing technologies, PCR- biochip technology etc..PCR is from nucleic acid water
Flat to detect to pathogen, entire experiment needs complete for 1~2 hour.When the major defect of this method is to carry out PCR detection
Need to rely on the real-time PCR and other a variety of corollary equipments and special PCR Lab and specially of PCR instrument or valuableness
Industry operator.PCR detection cannot achieve real-time test, the diagnosis of bed side and the scene application without special laboratory testing conditions,
Therefore be unable to satisfy base, user terminal, scene inspection demand.Meanwhile PCR detection there may be false positive and sensitivity not
The problems such as sufficient.
CRISPR/Cas system can identify exogenous DNA or RNA, and they are cut off, the expression of silencing foreign gene.
Just because of this accurate target function, CRISPR/Cas system is developed to a kind of efficient gene editing tool, can be with
The accurate edits that gene is pinpointed.Participation of the CRISPR/Cas in guide RNA (guide RNA, gRNA) and Cas9 albumen
Under, cell genomic dna to be edited will be counted as virus or exogenous DNA, accurately be sheared.The application of CRISPR/Cas9
There are some restrictive conditions.Firstly, the PAM sequence (NGG) that areas adjacent to be edited needs to have relatively conservative.Secondly, guide
RNA will be with the series complementary pairing of the upstream PAM.Furthermore 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
Specific knockdown of the CRISPR-Cas9 to target gene；Therefore it can design, prepare accuracy and selectively targeted target base
The key technology of the gRNA and CRISPR-Cas9 gene knockout of cause.
2015, a kind of completely new the second class CRISPR-Cas system-V-type system was found, the effect egg in the system
It is white to be named as Cpf1/Cas12a.Entitled " the Cpf1is a that Zhang Feng team delivers on November 22nd, 2015 at " Cell "
The article of 2 CRISPR-Cas system " of single RNA-guided endonuclease ofa Class.The system base
This workflow is similar with CRISPR/Cas9, or beats by " blacklist " system of CRISPR sequence invader
It hits.But the mode that gRNA is formed is different from CRISPR/Cas9 system: Cpf1/Cas12a albumen can be multiple with immature gRNA
It closes, and gRNA is processed, gRNA can then hybridize with the complementary region near PAM.Finally, external source double-stranded DNA (double
Strand DNA, dsDNA) it can be sheared, gene expression can be also silenced.However, while cutting target dsDNA, activation
Cpf1/Cas12a can also degrade target dsDNA neighbouring single stranded DNA (single strand DNA, ssDNA), referred to as
" attached cutting ", this activity are the key features of detection of nucleic acids platform newly developed.On April 27th, 2018, Doudna team
Two entitled " Two distinct RNase activities of have been delivered at " Science " simultaneously with Zhang Feng team
CRISPR-C2c2 enable guide-RNA processing and RNA detection " and " Multiplexed and
The paper of portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 ".Table
While cutting target dsDNA, can also degrade bright Cpf1/Cas12a target dsDNA neighbouring ssDNA.The two independent experiments
The V-type CRISPR system of targeting dsDNA has been transformed in room respectively, has become quick, cheap and super-sensitive diagnostic tool.
This discovery is expected to bring the influence for the property changed for scientific research and global public health.Utilize this new CRISPR skill
Art: CRISPR-Cpf1/Cas12a, highly sensitive can detect includes that zika virus infects, including dengue virus infection etc.
Disease, principle are by CRISPR-Cpf1/Cas12a in conjunction with isothermal nucleic acid amplification, detect the RNA or DNA of specificity.Separately
Outside, the report ssDNA of fluorescence is issued when which also includes a cutting.When Cpf1/Cas12a detects target dsDNA sequence
When column, ssDNase activity will cut report ssDNA, discharge detectable fluorescence signal.Both technologies are combined
New system can with extremely low concentration detect list RNA and list DNA molecular, have a good application prospect.According further to Zhang Fengtuan
The result of study of team shows that of the same clan, Cpf1/Cas12a albumen has biggish difference, and certain albumen of the same clan are inactive.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies of existing CRISPR/Cas system, provide a kind of with DNA
The protein component ScCas12a albumen of the novel C RISPR/Cas12a system of cleavage activity can be applied to specific nucleic acid detection.
The application that the object of the present invention is to provide ScCas12a albumen in terms of the cutting DNA and in terms of detection of nucleic acids.
Another object of the present invention is to provide a kind of nucleic acid detection system based on ScCas12a albumen.
Still a further object of the present invention is to provide a kind of nucleic acid detection method based on novel C RISPR/ScCas12a system.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Exploratory development of the present invention has found a kind of novel C as12a albumen with DNA cleavage activity, i.e. ScCas12a egg
White, nucleotide sequence is as shown in SEQ ID NO.1.
ScCas12a albumen has DNA cleavage activity, can be used as novel C RISPR/ScCas12a system and examines applied to nucleic acid
Survey, gene editing, gene modification, for based on Cas12a verification Molecular Detection and gene editing and modification provide a kind of necessity
The new selection of tool.
Therefore, applying below should all be within the scope of the present invention:
Application of the ScCas12a albumen in terms of cutting DNA.
ScCas12a albumen as or application in terms of preparing DNA cutting tool.
Application of the ScCas12a albumen in terms of detection of nucleic acids.
Application of the ScCas12a albumen in terms of the detection of nucleic acids based on CRISPR/Cas12a.
ScCas12a albumen as or preparation detection of nucleic acids tool in terms of application.
ScCas12a albumen as or preparation the detection of nucleic acids tool based on CRISPR/Cas12a in terms of application.
The ScCas12a albumen, which has, targets the work that external DNA sequence dna or internal genome sequence are specifically cut
Property, the DNA cleavage activity of specifically gRNA mediation.
It is additionally based on above-mentioned application, the present invention also provides a kind of nucleic acid detection systems based on CRISPR/Cas12a, including
ScCas12a albumen and gRNA.
Wherein, the design principle of the gRNA are as follows: when choosing gRNA targeting sequence, targeting sequence 5 ' end should have 5 '-
TTTN-3 ' sequence, and target sequence itself and do not formed between stable secondary structure, targeting sequence and remaining sequence not formed and stablize two
Level structure.
As a kind of selectable case, when target sequence is as shown in SEQ ID NO.2, the sequence of gRNA such as SEQ ID
Shown in NO.3.
A kind of nucleic acid detection method based on any system of claims 1 to 3 is to utilize ScCas12a albumen and right
The gRNA of target sequence is answered to carry out CRISPR detection of nucleic acids.
Specifically, the nucleic acid detection method based on CRISPR/Cas12a are as follows: by determined nucleic acid sample, SsCas12a
Albumen, gRNA, non-specific single-stranded fluorescence probe and reaction required buffer liquid are mixed into reaction system, carry out detection reaction.Specifically
It is that reaction system is placed in fluorescence analyser (BioTek) progress fluorescence analysis, with excitation wavelength 530nm/ launch wavelength 580nm reading
Take reacting hole fluorescent value.
Preferably, detection architecture includes: 2 μ l RPA products, and 45nM SsCas12a, 22.5nM gRNA, 100nM are non-specific
Single stranded DNA fluorescence probe and nuclease detect buffer.
The each component of the buffer is in the final concentration of of detection architecture: 20mM Tris, 60mM NaCl, 10mM
MgCl2,pH 7.3。
Reaction condition are as follows: reacted 1-3 hours at 37 DEG C.
Meanwhile in above-mentioned application and detection system, in addition to ScCas12a albumen itself of the present invention, function becomes
Body or its homologue or ortholog are all possible to retain part or all protein activity, the i.e. function of ScCas12a albumen
The application of variant or its homologue or ortholog, also should be within the scope of the present invention.
The functional variety may include that ScCas12a mutant (can be insertion, the mutation of missing or replacement sequence
Body), polymorphic etc..The functional variety also include ScCas12a albumen and another usually incoherent nucleic acid, protein or
The fusion product of polypeptide.Functional variety can be naturally occurring, be also possible to artificial.
In addition, based on above-mentioned technical proposal of the invention following content should be covered:
(1) present invention discharges fluorescence by the reporter dna chain of the attached cutting belt fluorophor of ScCas12a and the pure group that goes out
Then reaction system is placed in fluorescence analyser and detects nucleic acid product by group.Except detection of nucleic acids signal report of the present invention
Announcement method realizes the side of signal detection after the present invention can also generate attached cutting effect using activation ScCas12a by other
Case detects one or more target molecules present in sample.
(2) scheme described in (1), including based on the attached cutting belt biotin of ScCas12a, fluorophor, digoxin or
After the nucleic acid fragment of other labels, realize that nucleic acid product detects by colloidal gold lateral chromatography mode.The present invention can also pass through
The colloid gold particle of the attached cutting aggregationization of ScCas12a, makes colloid gold particle color change, to detect nucleic acid product signal.
(3) for the present invention in other specific nucleic acid detection schemes, one or more guide RNAs are designed to targeting one
The target molecule of kind or a variety of states that diagnose the illness.The disease can be human diseases, Animal diseases, plant disease；
(4) according to (3), the human diseases can be human infectious disease, cancer, organ disease, hematologic disease,
Disease of immune system, brain and the nervous system disease, endocrine system disease, genetic disease.
(5) according to (4), the human infectious disease can be and be caused by virus, bacterium or fungi.It can be breathing
Road syncytial virus, influenza A virus, influenza B virus, seasonal current Influenza Virus, parainfluenza virus, adenovirus, people's rhinopathy
Poison, human metapneumovirus, mumps virus, mycoplasma pneumoniae, chlamydia pneumoniae, mycobacterium tuberculosis, Middle East respiration syndrome hat
Shape virus, Bordetella pertussis, bacillus legionnaires,pneumophila, A streptococcus；It is former to can be human immunodeficiency virus, gonococcus, trachoma clothing
Body, ureaplasma urealyticum, human papilloma virus, microspironema pallidum, herpes simplex virus, human parvovirus；It can be hepatitis A
Virus, hepatitis type B virus, Hepatitis C Virus, Hepatitis D virus, Hepatitis E virus；Can be human cytomegalovirus,
Herpes virus hominis, Coxsackie virus, enterovirus EV 71/CA16, dengue fever virus, salmonella, Shigella, pylorus spiral
Bacillus, norovirus, intestinal adenovirus, rotavirus, Ebola virus.Dengue fever virus.
(6) according to (4), cancer can be lung cancer, colorectal cancer, gastric cancer, gastrointestinal stromal tumor, breast cancer, oophoroma, forefront
Gland cancer, thyroid cancer, cancer of pancreas, lymthoma etc..
(7) according to (4), hematologic disease and genetic disease may is that thalassemia, hemophilia, sickle cell are poor
Blood, Rett syndrome, cystic fibrosis, Huntington disease, fragile X mental retardation, Patau syndrome, Edwards syndrome, 21 3
Body syndrome, inherited metabolic disease, hereditary hearing impairment, Polycystic Kidney, congenital disorders of glycosylation, G6PD deficiency disease, benzene
Acetonuria, tyrosinemia, hepatolenticular degeneration, albinism, glycogenic thesaurismosis, hereditary breast cancer, inherited ovarian,
Heredity colorectal cancer etc.；
(8) according to (4), the organ disease, disease of immune system, brain and the nervous system disease, endocrine system disease can be with
It is cerebral apoplexy, hypertension, coronary heart disease, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, anaphylactia, class wind
Wet, multiple sclerosis, hereditary allergic dermatitis, diabetes, macular degeneration, ankylosing spondylitis etc..
(9) according to (3), the Animal diseases may is that Porcine epidemic diarrhea virus, porcine rotavirus A groups, pig transmissible
Marcy agent, aftosa, blue otopathy, swine fever, pig circular ring virus, African swine fever, porcine pseudorabies virus, pig Japanese B brain
Inflammation, pig parvoviral, swine flu, blue otopathy, Streptococcus suis, bacillus rhusiopathiae suis, rinderpest, PPR virus, sheep pox disease
Poison, pasteurella multocida, bird flu, newcastle disease virus, duck plague virus, chicken Marek's disease virus, infections chicken cloacal bursa
Virus, chlamydia felis, cat coronavirus, mycoplasma felis, feline infectious peritonitis, feline calicivirus, feline herpetovirus, cat whiting
Cytopenia, mycoplasma canis, hepatitis infectiosa canis virus, dog parainfluenza, dog Flu-A, canine parvovirus, canine distemper virus, dog are coronal
Virus, rabies viruses, Bartonella, toxoplasma, Leptospira, Babesia, Brucella, prawn infectivity muscle necrosis
Virus washes in a pan to shrimp cream head disease virus, prawn and draws syndrome virus, prawn infectious subcutaneous and haematopoietic necrosis virus, anthrax
Bacillus etc..
(10) in other specific nucleic acid detection schemes, one or more guide RNAs are designed to target the present invention
One or more microbial resistance genes.The resistant gene can be tetracycline resistant, aminoglycoside medicaments drug resistance, resistance to
Disinfectant, erythromycin-resistant, macrolide outlet, drug resistance of vancomycin, multidrug resistance efflux pump, mupirocin drug resistance, sulfanilamide (SN)
Class drug resistance, Tylosin drug resistance, fluoquinolone drug resistance, beta betaLactamases drug resistance, cephalosporin drug resistance, carbapenem enzyme are resistance to
Medicine, staphylococcus aureus resistance, chloramphenicol acyl transferase gene, bleomycin gene, puromycin gene, kanamycins
Gene, ammonia benzyl mycin gene, extended spectrumβ-lactamase drug resistant gene etc..
(11) in other specific nucleic acid detection schemes, one or more guide RNAs are designed to target the present invention
The target molecule of one or more idiotypes.The idiotype can be human single nucleotide's polymorphism and genotype,
Animal genotypes, plant genotype etc..
(12) according to (11), human single nucleotide's polymorphism and genotype can be disease-related polymorphisms site,
Including VKORC1, CYP2C9, CYP2C19 etc.；It can be character related polymorphic site, including lactose tolerance gene, caffeine
Metabolism, alcohol metabolism, skin is anti-oxidant, sense of taste susceptibility, alopecia etc.；It can be human leukocyte antigen (HLA)；
(13) according to (11), the animal genotypes, plant genotype, can be single nucleotide polymorphism, allele,
Breeding identification, transgenosis identification etc..
(14) in other specific nucleic acid detection schemes, one or more guide RNAs are designed to target the present invention
The target molecule of one or more detection environmental samples states.The environmental sample comes from food samples, drink sample, paper table
Face, fabric surface, metal surface, wood surface, frosting, pedotheque, water sample, atmosphere or other gaseous samples, or
A combination thereof.The detection environmental samples state can be virus, and bacterium, all kinds of microbial nucleic acids existences such as fungi can also
To be animal, Plant Genome derived nucleic acid existence is also possible to transgenic nucleic acid existence；
(15) for the present invention in other specific nucleic acid detection schemes, one or more sample types can be used for nucleic acid inspection
It surveys.The sample type can be tissue samples, saliva, blood, blood plasma, serum, excrement, urine, sputum, mucus, lymph
The swab of liquid, synovia, cerebrospinal fluid, ascites, pleural effusion, seroma, purulence or skin or mucomembranous surface, washing liquid etc.；
(16) for the present invention in other specific nucleic acid detection schemes, which can be carried on different substrates
On；The matrix can be test tube, drop, Solid Cavity road, micropore, particular substrate (such as paper substrate).
The invention has the following advantages:
The research of the invention finds that a kind of novel ScCas12a albumen with DNA cleavage activity, can be used as novel C RISPR/
ScCas12a system is applied to detection of nucleic acids, gene editing and modification, provides one for the nucleic acid molecules detection based on Cas12a
The new selection of kind essential tool.
And the nucleic acid detection system based on ScCas12a albumen can be in 25-37 DEG C of highly sensitive, the high-precision molecule of realization
Detection has specificity well and compatibility, and detection sensitivity is high, and testing cost is cheap, easy to operate, quick, application
Range is wide, has a good application prospect in terms of detection of nucleic acids.
Detailed description of the invention
Fig. 1 is ScCas12a segment PCR amplification result.
Fig. 2 is recombinant plasmid pET28a-ScCas12a carrier digestion qualification result.
Fig. 3 is ScCas12a protein expression result.
Fig. 4 is ScCas12a protein purification result.
Fig. 5 is ScCas12a Activity determination result；1, negative control；2, experimental group.
Fig. 6 is ScCas12a gRNA purification result.
Fig. 7 is the detection of nucleic acids result based on ScCas12a.
Fig. 8 is ScCas12a nucleic acid detecting sensitivity testing result (black LbCas12a, grey ScCas12a).
Fig. 9 is ScCas12a detection of nucleic acids specific detection result.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.To those skilled in the art, other any without departing from Spirit Essence and original of the invention
Changes, modifications, substitutions, combinations, simplifications made by reason is lower, should be equivalent substitute mode, are included in protection of the invention
Within the scope of.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Unless otherwise indicated, the present invention uses immunology, biochemistry, chemistry, molecular biology, microbiology, thin
Born of the same parents' biology, genomics and recombinant DNA etc. are the conventional technical ability of this field.Referring to Pehanorm Brooker (Sambrook), not in
Odd (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 laboratory manual " (CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY) (F.M. Austria Su Beier (F.M.Ausubel) et al. editor, (1987))；" Enzymology method "
(METHODS IN ENZYMOLOGY) series (Academic Press Inc): " PCR2: practical approach " (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)).
Isopropyl vulcanization-D- galactoside (IPTG) used in as the following examples is bought from Sigma company.Ni
Sepharose FF is bought from GE Healthcare.Protein purification consumptive material is bought from green skies company.4 30kDa of Amicon is super
Chimney filter is bought from Millipore company.Phusion archaeal dna polymerase, FastDigestNotI, FastDigestAscI inscribe
Enzyme, T4 ligase are bought from Thermo company.PCR clean up and gel reclaims kit are purchased from Qiagen company.
The discovery of 1 Cpf1/ScCas12a gene of embodiment
We have found a kind of Cas12a albumen with DNA cleavage activity from Smithella sp.SC_K08D17, are denoted as
ScCas12a (sequence is as shown in SEQ ID NO.1).DsRNA substrate, ScCas12a albumen and gRNA are added in vitro anti-
System is answered, discovery dsRNA substrate can specifically be cut under gRNA mediation by ScCas12a albumen.
Meanwhile the present inventor team, the study found that when choosing gRNA targeting sequence, targeting sequence 5 ' end should have 5 '-
TTTN-3 ' sequence, and target sequence itself and do not formed between stable secondary structure, targeting sequence and remaining sequence not formed and stablize two
Level structure, under this sgRNA design principle, ScCas12a, which has, targets external DNA sequence dna or internal genome sequence progress spy
The activity of different cutting.
Following embodiment gives the preparation and active confirmatory experiment case of ScCas12a albumen.
The clone of 2 Cpf1/ScCas12a gene of embodiment and protein expression
1, PCR amplification Cas12a sequence
(1) design primer
It is as follows according to ScCas12a sequence design upstream and downstream primer, sequence:
Upstream primer (shown in SEQ ID NO.4):
GTGCGGCCGCCATGCAGACCCTGTTTGAGAACTTCACA；
Downstream primer (shown in SEQ ID NO.5):
GTGGCGCGCCTGGCATAGTCGGGGACATCATATG。
(2) PCR amplification
Using above-mentioned upstream and downstream primer, using high-fidelity DNA polymerase (phusion archaeal dna polymerase) in different annealing
At a temperature of to target fragment carry out PCR amplification.As a result as shown in Fig. 1, PCR purpose band (about 4000bp).
2, construction recombination plasmid pET-28a-ScCas12a
(1) pcr amplification product purifies: purification kit (the Clean up examination that product passes through Qiagen company after PCR amplification
Agent box) carry out purification process；
(2) using the quick restriction enzyme NotI (FastDigestNotI) of Thermo company and NotI
(FastDigestAscI) double digestion is carried out；
(3) digestion products pass through micro-example gel reclaims kit (MiniElute) purification and recovery of Qiagen company；
(4) product of purification and recovery is connected to the pET28a-ccdB-CmR for also passing through the processing of NotI and AscI double digestion
On carrier, recombinant plasmid pET-28a-ScCas12a is obtained；
Wherein, pET-28-ccdB-CmR carrier used is the preservation of this laboratory, is with prokaryotic expression carrier pET28a (purchase
Managed in biological unifier) basis, it is engineered that NotI-ccdB-CmR-AscI sequence is added between HindIII and XhoI restriction enzyme site
Column, are made pET-28-ccdB-CmR carrier.
3, the identification of recombinant plasmid pET-28a-ScCas12a
To identify pET-28a-ScCas12a recombinant vector correctness, we are to recombinant plasmid pET-28a-ScCas12a
Carry out digestion identification and sequencing identification.
Digestion identification is carried out using Asc I or NotI single endonuclease digestion and Asc I or NotI double digestion respectively, as a result such as attached drawing 2
It is shown, the experimental results showed that, the digestion products size of all experimental groups is to be consistent with expection, thus can tentatively judge that we obtain
The carrier arrived is correct pET-28a-ScCas12a carrier.
In addition, sequencing result also indicates that ScCas12a sequence is correctly cloned into pET28a.
4, the prokaryotic expression of ScCas12a albumen
(1) it will identify that correct recombinant plasmid pET-28a-ScCas12a is converted to BL21 (DE3) expression bacterial strain (to be purchased from
Transgen company) in.Recombinant bacterium is obtained by positive identification.
(2) monoclonal of picking recombinant bacterium 37 DEG C of overnight incubations into 50mL LB culture medium.According to the inoculum concentration of 1:100,
Overnight strain, which is seeded in 1L LB culture medium 37 DEG C of cultures, adds IPTG to final concentration to OD600=0.6, ice-water bath 30min
For 0.5mM, continue to cultivate 4h in 15 DEG C.Thalline were collected by centrifugation saves in -80 DEG C.
5, it detects and optimizes ScCas12a protein expression
Recombinant plasmid pET-28a-ScCas12a is transferred in BL21 (DE3), is expressed at 37 DEG C with 0.2mM inducible protein,
And electrophoretic analysis is carried out to precipitating after cracking and supernatant.(as shown in Fig. 3).
The purifying of 3 ScCas12a albumen of embodiment
1, the purification process of ScCas12a albumen
Bacterium solution after inducing expression is centrifuged, and thallus is resuspended in lysis buffer, carries out ultrasonication (70% vibration
Width, 2s On/4s Off, 3 minutes, Sonics 750w Ultrasound Instrument), it is centrifugated supernatant.Extremely by protein cleavage supernatant loading
Ni Sepharose FF after balance washes away foreign protein to be greater than the lysis buffer of 30 times of bed volumes, with elution buffer
It is eluted, with Superdex 200,10/300 gel chromatographic columns of Tricorn are purified.SDS-PAGE points are carried out after elution
Analysis observation result and gel column purification, the Cas12a albumen after purification of acquisition.Wherein, lysis buffer includes 50mM
Tris-HCl, pH8.0 300mM NaCl, 5% glycerol, 20mM imidazoles.Elution buffer includes 50mM Tris-HCl, pH8.0
300mM NaCl, 5% glycerol, 250mM imidazoles.
Obtained albumen dilutes three times with 50mM Tris-HCl pH8.0 300mM NaCl5% glycerol, and super with 30kDa
Chimney filter concentration.After adding glycerol to final concentration 50%, packing liquid nitrogen flash freezer is stored in -80 DEG C.
2, ScCas12a protein purification result
Carry out large-scale purification again after optimized purification step, purpose band is about 170kDa.As shown in Fig. 4, it is seen that
The purity and yield of purifying are higher.
ScCas12a purification schemes of the present invention simplify TEV cutting labelling step, have substantially simplified purifying process and purifying
Cost, meanwhile, this method can ensure that protein active.
The Activity determination of 4 ScCas12a albumen of embodiment
1, it is the activity for detecting ScCas12a albumen, passes through the activity of the ScCas12a of experiment in vitro verifying purifying.
We have prepared the pUC19-B2 plasmid containing target sequence, and (plasmid origin is in article: CRISPR-Cas12a
Target binding unleashes single-stranded DNase activity) as inspection ScCas12a albumen
Active substrate, choose plasmid on a site design sgRNA, active ScCas12a albumen in conjunction with sgRNA after can
Targeting DNA is cut into two segments, so that it is determined that whether ScCas12a albumen is active.
The plasmid that specifically 100ng is linearized through SacI, the ScCas12a albumen of 20ng gRNA, 250ng after purification,
It mixes in 1 × NEB buffer3 final volume 10 μ L, 37 DEG C of incubation 60min, reaction is with the termination of 0.1 μ L Proteinase K, in 1% agar
Electrophoretic analysis ScCas12a albumen external activity is carried out in sugared gel.As a result as shown in Fig. 5, only when being added to ScCas12a
DNA just can be successfully cut into two segments after albumen, thus confirm we extract, the ScCas12a albumen that purifies in vitro
It is active.
The acquisition of gRNA described in experiment: design can identify the targeting sequence of plasmid: tttaGATCGTTACGCTAACTATG
A transcribes out sgRNA by T7 RNA polymerase.
Detection of nucleic acids of the embodiment 5 based on ScCas12a
1, target RNA is prepared
Target nucleotide can pass 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.
Recombinase polymeric enzymatic amplification RPA (Recombinase Polymerase Amplification): NCBI is used
Primer blast designs RPA primer, and amplified fragments size is 80-120nt, and the denaturation temperature of primer can be 54-67 DEG C, Opt
=60, length 30-35nt, Opt=32, G/C content is 40-60% in primer, according to implementation sequence synthetic DNA primer.
Template sequence:
atttcacacaggaaacagctatgaccatgattacgccaagcttGACGACAAAAtttaGATCGTTACGC
TAACTATGAgggctgtctgtgaatgctaggatccccgggtaccgagctcgaattcactggccgtcgttttacaacg
tcgtgactgggaaaaccctggcgttacccaacttaatcgcc
Primer sequence:
FP:AATTCTAATACGACTCACTATAGggccagtgaattcgagctcggtacccgggg atcc
RP:atttcACACAGGAAACAGCTATGACCATGATTACG
It refers to respectivelyBasic andBasicRT (TwistDx) kit carries out RPA reaction,
Unlike, before template segments addition, the MgAc of 280mM, i.e. magnesium acetate is first added.It is reacted 30 minutes at 37 DEG C.
Glue separation and purifying (using MinElute gel extraction kit (Qiagen) kit), after purification
DsDNA be incubated overnight (use T7 RNA polymerase (Thermo) kit) with 37 DEG C of T7 polymerase, then use
RNeasy mini kit (Qiagen) kits RNA, to obtain target nucleus RNA.
2, gRNA is prepared
GRNA primer sequence design principle: when choosing targeting sequence, targeting sequence 5 ' end should have 5 '-TTTN-3 ' sequence；
And targeting sequence itself does not form between stable secondary structure, targeting sequence and remaining sequence and does not form stable secondary structure.It can lead to
Cross http://www.rgenome.net/cas-designer/ online software Computer Aided Design.
Primer construction:
5 '-targeting sequence-" ATCTACACTTAGTAGAAATTA "-CCCTATAGTGAGTCGTATTACA-3 '
Wherein " ATCTACACTTAGTAGAAATTA " sequence can be replaced " ATCTACAATAGTAGAAATTA ".
Referring to T7 RNApolymerase kit (Thermo) kit specification, by the DNA fragmentation with T7 promoter, T7
Primer, the mixing of T7 polymerase, 37 DEG C of overnight incubations；RNeasy mini kit (Qiagen) is used again, obtains the gRNAs of purifying.
As shown in Figure 6.
T7 primer sequence: TGTAATACGACTCACTATAGGG
T7 gRNA primer sequence:
TCATAGTTAGCGTAACGATCATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTACA。
3, Cpf1/Cas12a Activity determination
Detection architecture includes: 100ng nucleic acid primer, the ScCas12a of 45nM purifying, 22.5nM gRNA and nuclease inspection
It surveys buffer (20mM Tris, 60mM NaCl, 10mM MgCl 2, pH 7.3).React 1h.1 μ l Proteinase K is added
(Thermo) reaction is terminated.Agarose gel electrophoresis analysis is as a result, such as Fig. 7.
4, detection of nucleic acids
Detection architecture includes: 2 μ l RPA products, the ScCas12a of 45nM purifying, 22.5nM gRNA, and 100nM exists
The reporter dna chain of ScCas12a capable of emitting fluorescence when cutting, i.e., non-specific single stranded DNA fluorescence probe (DNAseAlert QC
System Thermo Scientific), 0.5 μ l RNase inhibitor (Promega) and nuclease detect buffer (20mM
Tris,60mM NaCl,10mM MgCl2,pH 7.3)。
Reaction system is placed in fluorescence analyser (BioTek), reacts 1-3 hours under 37 DEG C (unless otherwise indicated), fluorescence
Dynamic testing 5 minutes primary.
Analysis SHERLOCK fluorescence data: the fluorescence data in order to calculate removal background facilitates the ratio between different condition
Compared with the initial fluorescence of sample is removed.Background fluorescence (no target nucleotide or without gRNA under conditions of) can be removed from sample, from
And obtain the data of background correction fluorescence.
For testing result as shown in figure 8, compared with reported LbCas12a, ScCas12a has same detection sensitivity.
Detection of nucleic acids specificity of the embodiment 6 based on ScCas12a
1, experimental method
By determined nucleic acid sample described in same embodiment 4, ScCas12a albumen, gRNA, non-specific single-stranded fluorescence probe
It is mixed into reaction system with reaction required buffer liquid addition, it is glimmering to read reacting hole with excitation wavelength 530nm/ launch wavelength 580nm
Light value.In the presence of target, compared with negative control sample, fluorescence signal can receive at 580nm.
According to SsCas12a for substrate and mutation Substrate fluorescence signal intensity difference judgement detection specificity.
Substrate sequence:
atatgGTGCCATGGACTTTAGTACATTGCAAGATACTAAATGTGAGGTAC
Mutant nucleotide sequence:
atatgGTGCCATGGACTTTAAAACATTGCAAGATACTAAATGTGAGGTAC
2, result has good as shown in figure 9, can distinguish to non-specific template that is special and non-fully matching
Verify detection specificity.
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 Cas12a gene, the building of recombinant expression carrier, the expression of Cas12a 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.
Sequence table
<110>the universal Li Hua Science and Technology Ltd. in Guangzhou
<120>application of the novel ScCas12a in terms of detection of nucleic acids
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3753
<212> DNA
<213>ScCas12a sequence (ScCas12a)
<400> 1
atgcaaacac tatttgaaaa tttcacaaat cagtatccgg tgagtaagac tttgcgtttt 60
gagttgatcc cgcagggaaa aacaaaagat tttatagaac agaaaggtct tttaaaaaaa 120
gacgaggatc gtgcggaaaa atataagaaa gtaaaaaaca ttattgatga atatcataaa 180
gatttcattg agaaatcact gaacggttta aaattagacg ggcttgagaa gtataagact 240
ttatatttaa agcaggaaaa agacgataaa gataaaaagg cgtttgataa agaaaaagaa 300
aatcttagga aacagattgc gaatgctttt agaaacaatg aaaagtttaa aacgctcttt 360
gccaaggaat taataaagaa cgatttgatg agttttgcct gtgaggaaga caaaaaaaac 420
gtaaaggagt ttgaagcttt tacaacttat ttcactgggt ttcaccagaa cagggcaaac 480
atgtatgtgg cggatgagaa acgaacggct attgcgagcc ggcttataca tgagaatctg 540
cccaagttca ttgataacat caagatattt gaaaagatga aaaaagaagc gcccgagctt 600
ctttcccctt ttaatcaaac attaaaggat atgaaggatg ttatcaaggg tacaacattg 660
gaagaaatat tttcactgga ttatttcaat aagacactta cacaatcggg gattgacata 720
tacaattccg taatcggcgg cagaacaccc gaagagggaa aaacaaaaat caagggctta 780
aacgaatata tcaatactga ttttaaccag aagcagaccg ataagaaaaa aagacagccc 840
aagttcaagc aactctacaa gcagattttg agcgacaggc agagcctgtc atttattgcg 900
gaggcattca aaaatgatac tgaaattctg gaagcaatag aaaaattcta tgtaaatgaa 960
ttgctgcatt tttccaatga aggcaaatca accaatgtat tggatgcaat caaaaatgct 1020
gtaagcaatc ttgaatcttt caacctgacc aagatgtatt tcagaagcgg cgcttcattg 1080
accgatgttt ccaggaaagt atttggagaa tggagcatta ttaacagggc tttagataat 1140
tactacgcaa caacctaccc catcaagcca agagagaaat cagagaaata cgaagagcgg 1200
aaagagaaat ggctcaaaca ggattttaac gtcagcctga ttcagaccgc aattgatgag 1260
tacgacaatg aaacagttaa gggaaaaaac agtgggaaag tcatagctga ttactttgcg 1320
aagttctgcg atgacaaaga gactgattta attcagaagg tcaatgaagg ctatatagct 1380
gtcaaggatt tgctgaacac cccttgtcct gaaaatgaga agctcgggag caataaggat 1440
caggtcaaac agataaaggc atttatggac agcataatgg acattatgca ttttgtaaga 1500
ccgctgagcc tgaaggatac tgataaagag aaagacgaaa cattctatag tctgtttacc 1560
cctctatacg accatctgac ccagaccatc gcactctaca ataaggtgcg taactacctc 1620
acacagaaac catacagcac ggaaaagata aaactgaact ttgaaaactc cacattgctg 1680
ggtggatggg atttaaataa agaaacagac aatacagcta ttatactgag gaaagataac 1740
ctttattact tgggcattat ggacaagaga cataacagaa tattcagaaa cgtacctaaa 1800
gcggataaaa aggatttctg ctacgagaaa atggtttaca agcttctgcc tggagcaaat 1860
aaaatgctgc cgaaggtatt cttttcgcag tcacggatac aggaatttac tccgtcagcc 1920
aaactgctgg aaaactacgc gaatgagacg cacaaaaaag gtgataattt caacctgaat 1980
cattgccata aattaattga ttttttcaaa gactcaatca acaaacacga ggactggaaa 2040
aactttgatt tcaggttttc agccacgtcc acctatgctg atttaagcgg attctatcac 2100
gaggtggaac atcagggcta taagataagt ttccagagcg tagccgattc attcattgac 2160
gatttggtca atgaagggaa actctatctt ttccaaatat acaacaagga tttctcacct 2220
ttcagtaagg gcaagcccaa cctgcacacc ctctactgga agatgctctt tgatgagaat 2280
aatttaaaag acgtggttta taaactaaat ggtgaggccg aggtattcta ccgcaagaaa 2340
tccattgccg agaaaaacac gaccattcat aaggccaatg aaagtattat taacaagaat 2400
ccagacaatc cgaaagccac aagtacattt aactacgaca tcgtcaagga taaacgctac 2460
accattgata agttccaatt ccacattccc ataaccatga atttcaaggc tgaaggcata 2520
ttcaatatga accagagggt caaccagttc ctcaaggcca acccagatat taatatcatc 2580
ggaatagaca ggggagaaag gcatctactt tactacgccc tgataaatca gaaaggtaag 2640
attctcaagc aggacacctt gaatgtcatt gccaatgaaa agcagaaagt tgactatcac 2700
aacctgctgg acaagaaaga gggtgataga gccacggcaa ggcaggaatg gggcgtaatt 2760
gagaccatta aggaactgaa ggaaggttat ctgtcgcagg tcatccacaa gctgaccgat 2820
ttgatgattg aaaacaacgc catcattgtg atggaagatt tgaacttcgg tttcaagcgt 2880
ggaaggcaga aggtggagaa gcaggtttac cagaagtttg agaaaatgct gattgataaa 2940
ctcaattacc ttgtggataa gaataaaaaa gcaaatgaac ttggcggtct gctcaacgca 3000
ttccagttag cgaacaagtt tgaaagtttc cagaaaatgg ggaagcagaa cggatttatt 3060
ttctacgtgc ctgcgtggaa cacaagcaag accgatcctg ccacaggttt cattgatttc 3120
ctgaaaccca gatatgagaa cctgaaccag gcaaaggact tctttgagaa gtttgattcc 3180
atccgtctca acagcaaggc agattatttt gaatttgctt ttgattttaa aaacttcacc 3240
gaaaaggcag acggtggaag gacgaaatgg acagtttgca ccaccaatga ggacaggtac 3300
gcttggaaca gggcgttaaa caacaacagg ggcagtcagg aaaaatatga tatcacagca 3360
gaactgaaat ccctgtttga cggaaaggtg gactacaaaa gcggcaagga tttgaaacag 3420
cagatagcaa gtcaggaatc tgctgatttc tttaaggcat taatgaaaaa tttaagcatt 3480
accctttcat tgcggcacaa caacggagag aaaggagata atgagcagga ttatatttta 3540
tctcctgtag cagacagcaa gggaagattc tttgattcaa ggaaagcaga tgacgatatg 3600
cccaagaatg ccgacgccaa cggcgcttat catatcgcgc ttaaaggttt atggtgtctg 3660
gaacagatca gtaagacgga cgacctgaaa aaagtaaagt tagccataag caataaagag 3720
tggcttgaat ttgtgcaaac actaaaagga taa 3753
<210> 2
<211> 185
<212> DNA
<213>template target sequence (Target sequence)
<400> 2
atttcacaca ggaaacagct atgaccatga ttacgccaag cttgacgaca aaatttagat 60
cgttacgcta actatgaggg ctgtctgtga atgctaggat ccccgggtac cgagctcgaa 120
ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 180
tcgcc 185
<210> 3
<211> 40
<212> DNA
<213>gRNA sequence (gRNA)
<400> 3
taatttctac tattgtagat gatcgttacg ctaactatga 40
<210> 4
<211> 38
<212> DNA
<213>upstream primer (forward primer)
<400> 4
gtgcggccgc catgcagacc ctgtttgaga acttcaca 38
<210> 5
<211> 34
<212> DNA
<213>downstream primer (reverse primer)
<400> 5
gtggcgcgcc tggcatagtc ggggacatca tatg 34
<210> 6
<211> 3885
<212> DNA
<213>the ScCas12a protein gene sequence (Optimized ScCas12a) after optimizing
<400> 6
atgcagaccc tgtttgagaa cttcacaaat cagtacccag tgtccaagac cctgcgcttt 60
gagctgatcc cccagggcaa gacaaaggac ttcatcgagc agaagggcct gctgaagaag 120
gatgaggacc gggccgagaa gtataagaag gtgaagaaca tcatcgatga gtaccacaag 180
gacttcatcg agaagtctct gaatggcctg aagctggacg gcctggagga atacaagacc 240
ctgtatctga agcaggagaa ggacgataag gataagaagg cctttgacaa ggagaaggag 300
aacctgcgca agcagatcgc caatgccttc cggaacaatg agaagtttaa gacactgttc 360
gccaaggagc tgatcaagaa cgatctgatg tctttcgcct gcgaggagga caagaagaat 420
gtgaaggagt ttgaggcctt caccacatac ttcaccggct tccaccagaa ccgcgccaat 480
atgtacgtgg ccgatgagaa gagaacagcc atcgccagca ggctgatcca cgagaacctg 540
ccaaagttta tcgacaatat caagatcttc gagaagatga agaaggaggc ccccgagctg 600
ctgtctcctt tcaaccagac cctgaaggat atgaaggacg tgatcaaggg caccacactg 660
gaggagatct ttagcctgga ttatttcaac aagaccctga cacagagcgg catcgacatc 720
tacaattccg tgatcggcgg cagaacccct gaggagggca agacaaagat caagggcctg 780
aacgagtaca tcaataccga cttcaaccag aagcagacag acaagaagaa gcggcagcca 840
aagttcaagc agctgtataa gcagatcctg agcgataggc agagcctgtc ctttatcgcc 900
gaggccttca agaacgacac cgagatcctg gaggccatcg agaagtttta cgtgaatgag 960
ctgctgcact tcagcaatga gggcaagtcc acaaacgtgc tggacgccat caagaatgcc 1020
gtgtctaacc tggagagctt taacctgacc aagatctatt tccgctccgg cacctctctg 1080
acagacgtga gccggaaggt gtttggcgag tggagcatca tcaatagagc cctggacaac 1140
tactatgcca ccacatatcc aatcaagccc agagagaagt ctgagaagta cgaggagagg 1200
aaggagaagt ggctgaagca ggacttcaac gtgagcctga tccagaccgc catcgatgag 1260
tacgacaacg agacagtgaa gggcaagaac agcggcaaag tgatcgtcga ttattttgcc 1320
aagttctgcg acgataagga gacagacctg atccagaagg tgaacgaggg ctacatcgcc 1380
gtgaaggatc tgctgaatac accctgtcct gagaacgaga agctgggcag caataaggac 1440
caggtgaagc agatcaaggc ctttatggat tctatcatgg acatcatgca cttcgtgcgc 1500
cccctgagcc tgaaggatac cgacaaggag aaggatgaga cattctactc cctgttcaca 1560
cctctgtacg accacctgac ccagacaatc gccctgtata acaaggtgcg gaactatctg 1620
acccagaagc cttacagcac agagaagatc aagctgaact tcgagaacag caccctgctg 1680
ggcggctggg atctgaataa ggagacagac aacacagcca tcatcctgag gaaggaaaac 1740
ctgtactatc tgggcatcat ggacaagagg cacaatcgca tctttcggaa cgtgcccaag 1800
gccgataaga aggactcttg ctacgagaag atggtgtata agctgctgcc tggcgccaac 1860
aagatgctgc caaaggtgtt cttttctcag agcagaatcc aggagtttac cccttccgcc 1920
aagctgctgg agaactacga aaatgagaca cacaagaagg gcgataattt caacctgaat 1980
cactgtcacc agctgatcga tttctttaag gactctatca acaagcacga ggattggaag 2040
aatttcgact ttaggttcag cgccacctcc acctacgccg acctgagcgg cttttaccac 2100
gaggtggagc accagggcta caagatctct tttcagagca tcgccgattc cttcatcgac 2160
gatctggtga acgagggcaa gctgtacctg ttccagatct ataataagga cttttcccca 2220
ttctctaagg gcaagcccaa cctgcacacc ctgtactgga agatgctgtt tgatgagaac 2280
aatctgaagg acgtggtgta taagctgaat ggcgaggccg aggtgttcta ccgcaagaag 2340
agcattgccg agaagaacac cacaatccac aaggccaatg agtccatcat caacaagaat 2400
cctgataacc caaaggccac cagcaccttc aactatgata tcgtgaagga caagagatac 2460
accatcgaca agtttcagtt ccacatccca atcacaatga actttaaggc cgagggcatc 2520
ttcaacatga atcagagggt gaatcagttc ctgaaggcca atcccgatat caacatcatc 2580
ggcatcgaca gaggcgagag gcacctgctg tactatgccc tgatcaacca gaagggcaag 2640
atcctgaagc aggataccct gaatgtgatc gccaacgaga agcagaaggt ggactaccac 2700
aatctgctgg ataagaagga gggcgaccgc gcaaccgcaa ggcaggagtg gggcgtgatc 2760
gagacaatca aggagctgaa ggagggctat ctgtcccagg tcatccacaa gctgaccgat 2820
ctgatgatcg agaacaatgc catcatcgtg atggaggacc tgaactttgg cttcaagcgg 2880
ggcagacaga aggtggagaa gcaggtgtat cagaagtttg agaagatgct gatcgataag 2940
ctgaattacc tggtggacaa gaataagaag gcaaacgagc tgggaggcct gctgaacgca 3000
ttccagctgg ccaataagtt tgagtccttc cagaagatgg gcaagcagaa cggctttatc 3060
ttctacgtgc ccgcctggaa tacctctaag acagatcctg ccaccggctt tatcgacttc 3120
ctgaagcccc gctatgagaa cctgaatcag gccaaggatt tctttgagaa gtttgactct 3180
atccggctga acagcaaggc cgattacttt gagttcgcct ttgacttcaa gaatttcacc 3240
gagaaggccg atggcggcag aaccaagtgg acagtgtgca ccacaaacga ggacagatat 3300
gcctggaata gggccctgaa caataacagg ggcagccagg agaagtacga catcacagcc 3360
gagctgaagt ccctgttcga tggcaaggtg gactataagt ctggcaagga tctgaagcag 3420
cagatcgcca gccaggagtc cgccgacttc tttaaggccc tgatgaagaa cctgtccatc 3480
accctgtctc tgagacacaa taacggcgag aagggcgata atgagcagga ctacatcctg 3540
tcccctgtgg ccgattctaa gggccgcttc tttgactccc ggaaggccga cgatgacatg 3600
ccaaagaatg ccgacgccaa cggcgcctat cacatcgccc tgaagggcct gtggtgtctg 3660
gagcagatca gcaagaccga tgacctgaag aaggtgaagc tggccatctc caacaaggag 3720
tggctggagt tcgtgcagac actgaagggc aaaaggccgg cggccacgaa aaaggccggc 3780
caggcaaaaa agaaaaaggg atcctaccca tacgatgttc cagattacgc ttatccctac 3840
gacgtgcctg attatgcata cccatatgat gtccccgact atgcc 3885
Claims (13)
1. a kind of nucleic acid detection system based on CRISPR/Cas12a, which is characterized in that including ScCas12a albumen and gRNA.
2. nucleic acid detection system according to claim 1, which is characterized in that the design principle of the gRNA are as follows: choosing
When gRNA targets sequence, targeting sequence 5 ' end should have 5 '-TTTN-3 ' sequence, and target sequence itself and do not form stable second level
Stable secondary structure is not formed between structure, targeting sequence and remaining sequence.
3. nucleic acid detection system according to claim 2, which is characterized in that when target sequence is as shown in SEQ ID NO.2,
The sequence of gRNA is as shown in SEQ ID NO.3.
4. nucleic acid detection system according to claim 1, which is characterized in that the nucleotide sequence of ScCas12a albumen is such as
Shown in SEQ ID NO.1 or SEQ ID NO.6.
5. a kind of nucleic acid detection method based on any system of Claims 1 to 4, which is characterized in that utilize ScCas12a
Albumen and the gRNA of corresponding target sequence carry out CRISPR detection of nucleic acids.
6.ScCas12a application of the albumen in terms of cutting DNA.
7.ScCas12a albumen as or application in terms of preparing DNA cutting tool.
8.ScCas12a application of the albumen in terms of detection of nucleic acids.
Application of the 9.ScCas12a albumen in terms of the detection of nucleic acids based on CRISPR/Cas12a.
10.ScCas12a albumen as or preparation detection of nucleic acids tool in terms of application.
11.ScCas12a albumen as or preparation the detection of nucleic acids tool based on CRISPR/Cas12a in terms of application.
12. according to any application of claim 6~11, which is characterized in that ScCas12a albumen, which has, targets external DNA
The activity that sequence or internal genome sequence are specifically cut.
13. according to any application of claim 6~11, which is characterized in that there is ScCas12a albumen gRNA to mediate
DNA cleavage activity.
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