CN109468319A - For inhibiting HSV-1 to replicate and/or CRISPR/Cas9 system, method, kit and its application of target sequence expression - Google Patents

Abstract

The present invention provides for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed, comprising Cas9 albumen and gRNA, the coded sequence of the gRNA includes one of nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.6 or a variety of.The present invention also provides for inhibiting the composition, kit that HSV-1 is replicated and/or target sequence is expressed.The expression of HSV-1 virus replication, the target sequence for inhibiting HSV-1 virus can be effectively suppressed in the Cas9CRISPR/Cas9 system that the present invention uses, and applies also for the cancer for the treatment of HSV-1 virus infection or HSV-1 virus induction.

Description

For inhibit HSV-1 replicate and/or target sequence expression CRISPR/Cas9 system, Method, kit and its application

Technical field

The present invention relates to field of gene, are related to a kind of for inhibiting HSV-1 duplication and/or target sequence to express CRISPR/Cas9 system, method, kit and its application.

Background technique

Herpes simplex virus (Herpes simplex viruses, HSV) is the tunicary double-stranded DNA virus of tool, virus Genome includes 74 genes relevant to virus multiplication infection, can be divided into two kinds of serotypes HSV-1 and HSV-2.HSV-1 can be It is infected in crowd, wherein recurrent infection can occur for one third infection population.Herpes simplex keratitis is to lead to cornea Blind main reason, the annual whole world about 1,500,000 populations suffer from herpes simplex keratitis.

HSV-1 primary infection restrovirus may hide in nerves within the body cell, and the stimulated activation again of virus can occur Herpes simplex virus secondary infection.In secondary infection, latent virus is transported by the neuraxis to skin, and virus replication makes epithelium Cell death falls off, and forms new morbidity stove.Herpes simplex keratitis is that secondary infection causes on cornea by HSV-1 One of disease, be clinically most commonly seen one of viral keratitis, at the same be cause corneal blindness it is main because Element.The HSV-1 of latent infection conceals autogene group in nerve cell, only expresses incubation period correlation RNA (latency Associated transcript, LAT).Virus multiplication and the relevant albumen of infection are not expressed in incubation period, on the one hand to machine Body generates immunologic escape, and another aspect mainstream antiviral drugs also ineffective target spot cannot remove latent HSV-1.This leads Viral keratitis has been caused to be difficult to the problem of eradicating.

The regular short palindrome in cluster interval repeats system (clustered regularly interspaced short palindromic repeat；CRISPR-associated, CRISPR-Cas) be archaeal and bacterium resistance virus and plasmid The important immune defense system infected, for resisting the invasion of exogenous genetic material, such as phage virus and exogenous plasmid.Together When, it provides acquired immunity for bacterium: this is similar with the acquired immunity of mammal, when bacterium is by viral or outer When source plasmid is invaded, corresponding " memory " can be generated, so as to resist their invasion again.CRISPR/Cas system can be with It identifies exogenous DNA or RNA, and they is cut off, the expression of silencing foreign gene.

CRISPR-Cas system is divided into two major classes, and first major class CRISPR-Cas system is answered by the multi-subunit effect formed Object is closed to function；Second major class is functioned by single effect protein (such as Cas9, Cpf1, C2c1 etc.).Wherein, Cas9, Cpf1, C2c1 all have the DNA endonuclease activity of RNA mediation.

In nature, CRISPR/Cas system possesses plurality of classes, wherein CRISPR/Cas9 system be study it is most deep Enter, a kind of most mature classification of application.CRISPR-Cas9 is a kind of complex with endonuclease activity, is identified specific DNA sequence dna, carry out specific site cutting causes double-strand DNA cleavage (Double-strand breaks, DSB), in no mould Under conditions of plate, non-homogeneous recombination end connection (Non-homologous end joining, NHEJ) occurs, causes frameshit prominent Become (frameshift mutation), leads to gene knockout.CRISPR/Cas9 is after " zinc finger endonuclease (ZFN) ", " class The third generation " genome fixed point editing technique " occurred after activating transcription factor effector nuclease (TALEN) ".By feat of at The advantages that this is cheap, easy to operate, high-efficient, the worldwide laboratory rapidly CRISPR/Cas9, becomes biological scientific research Strong helper.

Nearly 2 years, research circle began trying to be applied to Cas9CRISPR/Cas9 system to inhibit HSV-1 duplication and/or target Sequence expression is marked, but since the practical application effect of gRNA is affected by many factors, can effectively be pressed down from viral genome level HSV-1 proliferation processed and the gRNA report of duplication are simultaneously few.

Therefore, it is necessary to provide a kind of for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed System, method, kit and its application.

Any manufacturer of any product in referenced herein or any document for being incorporated herein Specification, explanation, product specification and product table, be incorporated herein by reference, and can adopt in the practice of the invention With.More specifically, the document of all references is both incorporated herein by reference, and degree is as definite in each individual document Ground and individually indicating is incorporated by reference into herein.

Summary of the invention

To solve the above problems, the present invention provides a kind of for inhibiting HSV-1 duplication and/or target sequence to express CRISPR/Cas9 system, method, kit and its application.

First aspect present invention provides a kind of gRNA, and the coded sequence of the gRNA includes SEQ ID NO.1-SEQ ID One of nucleotide sequence shown in NO.6 is a variety of.

Second aspect of the present invention is provided for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed System includes Cas9 albumen and gRNA, and the coded sequence of the gRNA includes nucleotide shown in SEQ ID NO.1-SEQ ID NO.6 One of sequence is a variety of.

Preferably, in first aspect or second aspect, the gRNA be it is following one of 1) or 2):

It 1) include crRNA and tracrRNA, wherein the partial sequence complementarity of the partial sequence of crRNA and tracrRNA, simultaneously Form dimer, wherein crRNA includes boot sequence, and the coded sequence of the boot sequence includes SEQ ID NO.1-SEQ One of nucleotide sequence shown in ID NO.6 is a variety of；

2) the single-stranded sgRNA of mosaic type, wherein sgRNA is merged by crRNA with tracrRNA, and sgRNA includes guidance sequence Column, the coded sequence of the boot sequence include one of nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.6 or It is a variety of.

It will be appreciated by persons skilled in the art that gRNA is also wrapped in addition to comprising the boot sequence complementary with target DNA sequence Containing frame sequence.The form of gRNA can be the dimer being made of crRNA and tracrRNA, be also possible to it is artificial reconstructed by The single-stranded sgRNA of mosaic type made of crRNA and tracrRNA fusion.

It will be appreciated by persons skilled in the art that the RNA molecule of CRISPR RNA (crRNA) utilizes its a part of sequence Column (3 ' end) are incorporated in one by base pairing with the RNA molecule referred to as tracrRNA (trans-activating crRNA) It rises, forms chimeric RNA (i.e. tracrRNA/crRNA dimer), then, by another part sequence (5 ' end) and target of crRNA The site DNA carries out base pairing, and in this way, this chimeric RNA can guide the Cas9 protein binding to target DNA In sequence, specific site cutting is carried out.

It will be appreciated by persons skilled in the art that in practical application, tracrRNA and crRNA fusion can also be existed It is formed together and unidirectionally leads RNA (single guide RNA, sgRNA), i.e., the single-stranded sgRNA of mosaic type of the present invention is described The single-stranded sgRNA of mosaic type can guide in the Cas9 protein binding to target DNA sequence and carry out editing editor.

CRISPR system is from many different Bacteria Identifications and characterization, and any of these Cas9 enzymes can use In the method described in this article.It can be used for example from following any Cas9 albumen: corynebacterium (Corynebacter), Saudi Bordetella (Sutterella), Legionella (Legionella), treponema (Treponema), producing line Pseudomonas (Filifactor), Eubacterium (Eubacterium), streptococcus (Streptococcus), lactobacillus (Lactobacillus), mycoplasma (Mycoplasma), Bacteroides (Bacteroides), Flaviivola, Flavobacterium (Flavobacterium), Sphaerochaeta, Azospirillum (Azospirillum), gluconacetobacter category (Gluconacetobacter), eisseria (Neisseria), Ross Salmonella Belong to (Roseburia), Parvibaculum, staphylococcus (Staphylococcus), Nitratifractor, mycoplasma (Mycoplasma) and Campylobacter (Campylobacter).Several Cas9 expression vectors are available from Addgene.

In an embodiment of the present invention, using from micrococcus scarlatinae Cas9 (Streptococcus pyogenes Cas9, SpCas9), staphylococcus aureus Cas9 (Staphylococcus aureus Cas9, SaCas9), jejunum campylobacter bar Bacterium Cas9 (Campylobacter jejuni Cas9, CjCas9), streptococcus thermophilus Cas9 (Streptococcus Thermophilus Cas9, StCas9), diplococcus meningitidis Cas9 (Neisseria meningitidis Cas9, NmCas9), Lachnospira section Cpf1 (Lachnospiraceae Cpf1, LbCpf1), amino acid coccus Cpf1 The CRISPR system and Cas9 albumen of (Acidaminococcus Cpf1, AsCpf1) or other skilled in the art routinely manage The Cas9 albumen of solution.

Every kind of these albumen depends on different recognition site or PAM.For example, the PAM of Spy Cas9 is 5 '-NGG- That 3 ', Nme is 5 '-NNNNGATT-3 '.Spy uses 13 to 15 nucleotide target sequences.Nme and Sau uses 18 to 24 cores Thuja acid target sequence.

Those skilled in the art without departing from the principles of the embodiments of the present invention, can be to the code sequence of Cas9 albumen Column make suitable selection.

In an of the invention preferred embodiment, the amino acid sequence of the Cas9 albumen including but not limited to SEQ ID NO. 7Shown amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% consistent sequence.

In an embodiment of the present invention, the gRNA being made of boot sequence and frame sequence be can be used gRNA is corresponding DNA sequences encoding is cloned into carrier or transcribes in vitro.

In one embodiment of the invention, gRNA can be by complete complementary or substantially complementary or with certain complementing percentage, specifically Property identify the SA gene of the HSV-1 (in the embodiment of the present invention, " the SA gene of HSV-1 " and " target nucleotide sequences " can be with It exchanges).

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 Cas9 albumen Yu the target nucleotide sequences.In some embodiments, gRNA and its phase The complementarity between target nucleotide sequences answered 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, 23, on 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 the mature gRNA sequence of boot sequence and frame sequence composition.

In an embodiment of the present invention, the coded sequence of the boot sequence is SEQ ID NO.1-SEQ ID in table 1 One or more in nucleotide sequence shown in NO.6.

In embodiment of the present invention, what the gRNA frame sequence was routinely selected for those skilled in the art, the skeleton sequence Column can be the dimer being made of crRNA and tracrRNA (trans-activating crRNA), be also possible to manually change The single-stranded sgRNA of mosaic type as made of crRNA and tracrRNA fusion made.The frame sequence of gRNA can be conventional at present make Sequence the following is a kind of coded sequence of conventional frame sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC, Be also possible to some mutant constructed on this basis, these mutant and current conventional use of sequence have 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher similarity.

In embodiment of the present invention, the gRNA sequence can the target nucleotide of specific recognition may include and HSV-1 disease Sick related gene or polynucleotides.

" disease is related " gene or polynucleotides refer to compared with the tissue or cell of non-disease control, are deriving from disease Any gene or multicore glycosides of transcription or translation product are generated in the cell of the tissue of influence with abnormal level or with anomaly pattern Acid.In the expression of change situation relevant to the appearance of disease and/or progress, it can be one with abnormal high horizontal quilt The gene of expression；It can be the gene being expressed with abnormal low level.Disease related gene also refer to one or Multiple mutation are directly responsible for or are responsible for the unbalanced hereditary variations of etiologic etiological gene linkage of diseases with one or more Gene.Transcription or translation product can be known or unknown, and may be at normal or abnormal level.

In embodiments of the present invention, term " target nucleotide " refers to ribonucleotide or its analog." target nucleotide " It may include one or more modified nucleotide, such as nucleotide and nucleotide analog of methylation.

In an embodiment of the present invention, the Cas9 albumen further includes other joining peptides optionally, the company Connect peptide fragment specifically, including but not limited to epitope tag, reporter sequences and with it is following it is active one or more Protein domain: methyl enzymatic activity, hepatic Microsomal Aniline Hydroxylase, transcriptional activation activity, transcription repression activity, transcription releasing factor are living Property, histone modification activity, nuclear localization signal, 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 label, With thioredoxin (Trx) label.The example of reporter gene includes, but are not limited to glutathione-S-transferase (GST), horseradish Peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta galactosidase, β-glucuronidase, luciferase, It is green fluorescent protein (GFP), HcRed, DsRed, green fluorescin (CFP), yellow fluorescence protein (YFP), glimmering to include blue The autofluorescence albumen of photoprotein (BFP).

In an embodiment of the present invention, a kind of protein or egg can also be merged in the Cas9 fusion protein White matter segment, the protein or protein fragments combination DNA molecular combine 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 melt Close object and herpes simplex virus (HSV) BP16 protein fusions.In some embodiment of the invention, the fused protein For molecular labeling, it can be used to identify the position of target sequence using the Cas9 fusion protein of label.

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.

Second aspect of the present invention provides a kind of non-naturally occurring or engineering composition, and the composition includes one Kind or variety carrier, one or more carriers include component I and component II:

The component I includes the first regulating element, and the coding being operably connected with first regulating element is such as The coded sequence of Cas9 albumen described in second aspect；The component II includes the second regulating element, and is adjusted with described second The coded sequence of gRNA of the coding that section element is operably connected as described in second aspect；Wherein, component I and II is located at identical Or on different carriers.

In embodiments of the present invention, term " carrier " is preferably viral vectors, for example, retrovirus, replication defective Type retrovirus, adenovirus, replication-defective adenoviral and gland relevant viral vector.

In a preferred embodiment of the invention, it is related that the carrier is selected from retrovirus, slow virus, adenovirus or gland Virus.

In a preferred embodiment of the invention, the carrier is pLentiCRISPR V2 carrier.

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 (in an embodiment of the present invention, institute of the present invention " regulating element " stated can be understood as " expression casette ") include one or more pol III promoter (such as 1,2,3,4, Or more 5, pol III promoter), one or more pol II promoter (such as 1,2,3,4,5, or more pol II Promoter), one or more pol I promoter (such as 1,2,3,4,5, or more pol I promoter), or combinations thereof.This Outside, the first regulating element can also be T7 promoter, Sp6 promoter etc..The example of pol III promoter includes but is not limited to U6 With H1 promoter.The example of pol II promoter includes but is not limited to that reverse transcription Rous sarcoma virus (RSV) LTR promoter (is appointed Selection of land is with RSV enhancer), cytomegalovirus (CMV) promoter (optionally have cmv enhancer) is [see, e.g., wave sand Special (Boshart) et al., " cell " (Cell) 41:521-530 (1985)], SV40 promoter, dihyrofolate reductase starting Son, beta-actin promoter, phosphoglycerokinase (PGK) promoter and EF1 α promoter.

In some embodiments of the present invention, albumen coded sequence is through codon optimization, so as to for example true in specific cell It is expressed in nucleus.These eukaryocytes can be those of particular organisms or derive from particular organisms, such as mammal, including But it is not limited to people, mouse, rat, rabbit, dog or non-human primate.In general, codon optimization refers to by being used in place The codon more frequently or most frequently used in the gene of chief cell replaces at least one codon (example of native sequences Such as from about or more than about 1,2,3,4,5,10,15,20,25,50, or more codon maintain the natural acid sequence simultaneously And a nucleic acid sequence is modified to enhance the method for the expression in host cell interested.Different species are for having spy The certain codons for determining amino acid show specific preference.Codon preference (the difference that the codon between biology uses It is different) it is often related to the translation efficiency of mRNA (mRNA), and the translation efficiency is then considered dependent on (except other things) quilt The availability of the property of the codon of translation and specific transfer RNA (tRNA) molecule.The advantage one of selected tRNA into the cell As reflect the codon for being used most frequently for peptide synthesis.Therefore, gene can be customized to based on codon optimization in given life Best gene expression in object.Codon usage table can be readily available, such as use database (" Codon in codon Usage Database ") in, 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 the specific ordered series of numbers of codon optimization so as to The computerized algorithm expressed in specific host cell be also it is available, as (Aptagen is public for gene manufacture (Gene Forge) Department；Chris Jacobs (Jacobus), PA) and it is available.In some embodiments, one in the sequence of coding CRISPR enzyme A or multiple codons (such as 1,2,3,4,5,10,15,20,25,50, or more or all codons) corresponding to pair In the most frequently used codon of specific amino acids.

In a preferred embodiment of this invention, second regulating element includes but is not limited to one or more pol II promoter (such as 1,2,3,4,5, or more pol II promoter), one or more pol I promoter (such as 1,2,3, 5, or more 4, pol I promoter), or combinations thereof, pol III promoter (including but not limited to U6, H1 promoter), T7 Promoter, Sp6 promoter etc..

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.

Further, the composition further includes carrier pharmaceutically.

Pharmaceutical composition contains pharmaceutically acceptable ingredient, such as excipient, carrier and/or stabilizer.This ingredient Any pharmaceutical agent to the harmful immune response of individual for receiving composition is not induced including itself, and it is not inappropriate Toxicity and be administered.Pharmaceutically acceptable ingredient includes but is not limited to liquid, such as water, physiological saline, glycerol and ethyl alcohol.Medicine Acceptable salt is included therein on, such as inorganic acid salt, such as hydrochloride, hydrobromate, phosphate, sulfate etc.；With And acylate, such as acetate, propionate, malonate, benzoic acid salt etc..In addition, auxiliary substance such as soaks or emulsifier, PH buffer substance etc. can also exist in this carrier.The ingredient of other examples include lactose, sucrose, calcium phosphate, gelatin, Dextran, agar, pectin, peanut oil, sesame oil and water.The selection of carrier is not limitation of the invention.Optionally, the disclosure Composition in addition to rAAV carrier and other ingredients, may include other conventional medicine components, such as preservative, chemical stabilization Agent etc..Suitable exemplary compositions include microcrystalline cellulose, sodium carboxymethylcellulose, polysorbate80, benzyl carbinol, neoprene It is alcohol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, metagin, ethyl vanillin, glycerol, phenol, right Chlorophenol, gelatin and albumin.Pharmaceutically acceptable excipient is thoroughly discussed in REMINGTON'S It can get in PHARMACEUTICAL SCIENCES (Mack Pub.Co., N.J.1991).

Fourth aspect present invention provides a kind of for inhibiting HSV-1 duplication and/or target sequence in body cell or human body The method that list reaches, which comprises to allow to deliver and/or generate a effective amount of gRNA and/or Cas9 egg in cell Premised on white, deliver described in gRNA described in first aspect, CRISPR/Cas9 system or the third aspect described in second aspect Composition keeps the CRISPR/Cas9 system or composition sequence relevant with people's HBB mutated gene close and/or contacts.

Term " effective quantity " or " therapeutically effective amount ", which refer to, is enough to generate beneficial or desired biology and/or clinical effectiveness Amount.Therapeutically effective amount will change, this depend on compound, preparation or composition, disease and its seriousness and age, weight, The reactivity of physiological status and subject to be treated.When for applying to people or non-human mammal patient, recombinant vector is preferred (carrier being physiologically compatible with) is suspended in pharmaceutically acceptable delivery vector.Those skilled in the art are easy to selection and close Suitable carrier, and selected administration method can be depended on.

In one embodiment of the invention, the present invention provides a kind of methods for treating subject's virus infection comprising to by Examination person applies gRNA described in a effective amount of first aspect, CRISPR/Cas9 system described in second aspect or third aspect institute The composition stated, to treat virus infection.

In one embodiment of the present invention, application effective quantity is the quantity for reducing the duplication of DNA virus in subject.

In one embodiment of the present invention, application effective quantity is to reduce the encoded gene of target sequence in subject to produce The quantity of the expression of object.

In other factors, suitable dosage (effective quantity) will depend on type (such as the serotype of the carrier of selection Deng), application approach, mammal to be treated (such as people or non-human primates or other mammals), the age, weight, The mode of the general state and application of gender and subject to be treated.Therefore, suitable dosage can be different between patient.Ability Field technique personnel can be easily determined suitable effective quantity.

Dosage treatment can be single dose schedule or multi-dose scheme.Furthermore, it is possible to suitable many to subject's application Dosage.Those skilled in the art are easily determined the suitable quantity of dosage.However, it is contemplated that optional administration method or flat Weigh the benefit and any side effect treated, it may be necessary to adjust dosage.Depending on the treatment use of used recombinant vector, this Kind dosage can change.

With enough amount administered recombinant carriers to enter desired cell, and guarantee the function of transferred nucleic acid compositions Property enough levels, with provide the technical staff without unsuitable unfavorable treatment benefit or medical domain it is confirmable Medically acceptable physiological effect.

In embodiments of the present invention, term " makes the SA base of the CRISPR/Cas9 system or composition and HSV-1 Because relevant sequence is close and/or contacts " refer to component is delivered in vitro (in vitro) or (in vivo) environment in vivo In, the reaction solution of ex vivo environment such as those skilled in the art demand configuration according to specific experiments, vivo environment is such as intracellular； The term " close and/or contact " refers in vitro (in vitro) or in vivo (in vivo) environment, and each component can be with Sequence relevant with people's HBB mutated gene contacts, and under certain conditions occur those skilled in the art be anticipated that it is anti- It answers.

In an embodiment of the present invention, the present invention provides following methods, including deliver one into body cell or human body A or multiple polynucleotides, one or more carriers, one or more transcripts, and/or the albumen of one or more transcriptions.

In an embodiment of the present invention, the CRISPR/Cas9 system complexities combined with gRNA are delivered to body cell Or in human body.Conventional viral and non-viral base gene transfer method can be used by gRNA described in first aspect, second Composition described in CRISPR/Cas9 system or the third aspect described in aspect is introduced into the body cell or individual of people.

Cell of such method into cell culture or individual can be used and give gRNA described in first aspect, Composition described in CRISPR/Cas9 system or the third aspect described in two aspects.Non-virus carrier delivery system includes DNA matter Grain, RNA (such as transcript of carrier described herein), albumen, naked nucleic acid and compound with Delivery excipient (such as liposome) Nucleic acid.Viral vector delivery system includes DNA and RNA virus, they have sequestered or integration after being delivered to cell Type genome.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) is " raw Object technology " (1988) (Biotechnology) 6 (10): 1149-1154；Wei Nie (Vigne) " restores neurology and neurology department Learn " (Restorative Neurology and Neurosciece) 8:35-36 (1995)；Cray is write from memory (Kremer) & Perry section De Te (Perricaudet), 51 (1) " British Medical bulletin " (British Medical Bulletin): 31-44 (1995)； Clatter clatter (Haddada) et al. is breathed out, in " microbiology and immunology current topic " (Current Topics in Microbiologyand Immunology) in Dorr husband strangle (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, electricity turn, particle gun, virion, Liposome, immunoliposome, polycation or lipid: the reagent enhancing of nucleic acid conjugate, naked DNA, artificial virions and DNA Property intake.Fat transfection is described in such as U.S. Patent number 5,049,386,4,946,787 and 4,897,355 and fat transfection is tried Agent is commercially available (for example, TransfectamTM and LipofectinTM).Effective Receptor recognition rouge suitable for polynucleotides turns The cation and neutral lipid of dye include Felgner (Fil Ge Na), WO 91/17424；Those of WO 91/16024.Delivering It can be for cell (such as external or give 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 and 4,946,787)。

In a preferred embodiment of the invention, gRNA described in first aspect is first prepared, described in second aspect Carrier for expression of eukaryon, prokaryotic expression carrier or the in-vitro transcription carrier of CRISPR/Cas9 system；Then by carrier for expression of eukaryon Amplification, and prokaryotic expression carrier (or is transferred in Escherichia coli and lures by the carrier for expression of eukaryon for preparing the transfection grade of endotoxin-free Lead expression Cas9 albumen), prepare the albumen of Cas9 albumen.The coding DNA of Cas9 albumen can also be cloned into comprising T7 or In the in-vitro transcription carrier of Sp6 promoter, by being transcribed in vitro, the mRNA of Cas9 albumen is prepared.

In a preferred embodiment of the invention, gRNA described in first aspect is prepared, gRNA can be directly synthesized, or The coded sequence of gRNA is cloned into the transcription vector (such as pDR274 comes from Addgene) comprising T7 or Sp6 promoter, or logical The modes such as PCR, annealing, synthesis are crossed directly in the front end of the corresponding coding DNA of gRNA plus T7 or Sp6 promoter, then pass through body The method of outer transcription prepares gRNA；Or the coded sequence of the gRNA is cloned into carrier for expression of eukaryon pGEM-T-U6- In gRNA carrier, the pGEM-T-U6-gRNA carrier is to be expanded from pX330 carrier (Addgene) by way of PCR PCR product is connected into pGEM-T carrier (Promega) in such a way that TA is cloned by the frame sequence of U6 promoter and gRNA In it is obtained.

In a preferred embodiment of the invention, by the DNA of Cas9 albumen described in second aspect, (transcript mRNA is turned over The albumen translated) with the expression vector of gRNA (gRNA of the gRNA or synthesis that perhaps transcribe) by including but is not limited to lipid Body transfection, virus infection, electricity turns and the modes such as microinjection are imported into the body cell or human body of people.

Further, the viral vectors that the virus infection uses includes but is not limited to retroviral vector, slow disease Poisonous carrier, adenovirus vector and adenovirus first close viral vectors etc..

In an embodiment of the present invention, cell includes but is not limited to bone marrow cell, candidate stem cell (HSC), embryo Stem cell (ESC), inducing pluripotent stem cells (iPSC) and hematopoietic progenitor cells.

In an embodiment of the present invention, cell is 293T cell or Vero cell.

In an embodiment of the present invention, gRNA, CRISPR/Cas9 system and/or composition and cell are approached And/or after contact, one of following effect or a variety of occurs:

1) gRNA the and Cas9 Protein cleavage target sequence；

2) expression of the encoded gene product of the target sequence of gRNA reduces；

3) target sequence of gRNA is changed；

4) HSV-1 not reproducible；

5) HSV-1 is eliminated from cell.

In an embodiment of the present invention, two or more gRNA are delivered, and more than one target sequence is cut.

In an embodiment of the present invention, gRNA, CRISPR/Cas9 system and/or composition and cell are connect Close and/or contact occurs in vitro or in vivo.

Fifth aspect present invention provides a kind of eukaryotic host cell, includes gRNA, second aspect described in first aspect Composition described in the CRISPR/Cas9 system or the third aspect.

Sixth aspect present invention provides a kind of kit, comprising described in gRNA, second aspect described in first aspect One of eukaryotic host cell that composition described in CRISPR/Cas9 system or the third aspect, the 5th aspect provide is more Kind.

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 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 CRISPR/ described in a kind of gRNA as described in relation to the first aspect, second aspect One of eukaryotic host cell that composition described in Cas9 system or the third aspect, the 5th aspect provide or it is a variety of, the 6th A kind of application of the kit that aspect provides in prevention, improvement, and/or treatment HSV-1 related disease.

In a preferred embodiment of the invention, seventh aspect present invention provides a kind of gRNA provided such as first aspect Sequence or CRISPR/Cas9 system, non-naturally occurring or engineering the composition of second aspect offer, fourth aspect provide One of eukaryotic host cell or a kind of kit for providing of a variety of, the 5th aspect inhibiting HSV-1 duplication and/or target Application in sequence expression.

Eighth aspect present invention provides CRISPR/ described in a kind of gRNA as described in relation to the first aspect, second aspect The eucaryon host that non-naturally occurring or engineering composition, the 5th aspect provide described in Cas9 system or the third aspect is thin Born of the same parents are applied by following one or more methods:

It 1) will be described in CRISPR/Cas9 system described in gRNA, second aspect described in first aspect or the third aspect The eukaryotic host cell that non-naturally occurring or engineering composition, the 5th aspect provide individually is applied；

It 2) will be described in CRISPR/Cas9 system described in gRNA, second aspect described in first aspect or the third aspect Non-naturally occurring or engineering composition, the eukaryotic host cell of the 5th aspect offer and operation, biological therapy are immunized and control One or more for the treatment of use in conjunction；

3) by CRISPR/ described in gRNA described in the first aspect, second aspect by the way of delivering in vivo The eucaryon host that non-naturally occurring or engineering composition, the 5th aspect provide described in Cas9 system or the third aspect is thin Born of the same parents are directly delivered to patient's body and treat；

4) in-vitro transfection technology is first passed through by CRISPR/Cas9 system described in gRNA, second aspect described in first aspect Non-naturally occurring or engineering composition described in system or the third aspect is mixed with host cell, then will contain first party GRNA described in face, the host cell of composition described in CRISPR/Cas9 system or the third aspect described in second aspect are defeated It returns patient's body and implements treatment；

5) the eukaryotic host cell input patient's body implementation that the 5th aspect provides is controlled.

Ninth aspect present invention provides a kind for the treatment of method of HSV-1 disease, comprising:

It 1) will be described in CRISPR/Cas9 system described in gRNA, second aspect described in first aspect or the third aspect The eukaryotic host cell that non-naturally occurring or engineering composition, the 5th aspect provide individually treats patient；

It 2) will be described in CRISPR/Cas9 system described in gRNA, second aspect described in first aspect or the third aspect Non-naturally occurring or engineering composition, the eukaryotic host cell of the 5th aspect offer and operation, biological therapy are immunized and control One or more for the treatment of, which is combined, treats patient；

3) by CRISPR/ described in gRNA described in the first aspect, second aspect by the way of delivering in vivo The eucaryon host that non-naturally occurring or engineering composition, the 5th aspect provide described in Cas9 system or the third aspect is thin Born of the same parents are directly delivered to patient's body and treat；

4) in-vitro transfection technology is first passed through by CRISPR/Cas9 system described in gRNA, second aspect described in first aspect Non-naturally occurring or engineering composition described in system or the third aspect is mixed with host cell, then will contain first party GRNA described in face, the host cell of composition described in CRISPR/Cas9 system or the third aspect described in second aspect are defeated It returns patient's body and implements treatment；

5) the eukaryotic host cell input patient's body that the 5th aspect provides is implemented into treatment.

The utility model has the advantages that

In the course of the research, inventor passes through a large amount of research and exploration discovery, and inhibition HSV-1 is proliferated and is replicated GRNA sequence and cutting target spot need cautiously to select and screen.The difference of target spot and specific gRNA sequence, will lead to inhibition The ability of HSV-1 has differences.The present invention can be effectively suppressed virus replication using Cas9CRISPR/Cas9 system, inhibit to come from The expression of the target sequence of virus has the cancer for being applied to treatment virus infection or virus induction.

Often be difficult to eradicate after HSV-1 infection is latent, the present invention provides slave viral genome level inhibit HSV-1 to increase The means grown and replicated, by cell model it has been observed that the gRNA sequence referred in our inventions effectively can inhibit HSV-1 to increase It grows and replicates, be hopeful to be applied to clinical treatment HSV-1 infection.

Particularly, in a preferred embodiment, the gRNA sequence for targeting HSV-1 is inserted by the present invention PLentiCRISPR V2 carrier, by the way that the above-mentioned carrier of HEK293T cell line corotation and slow virus package carrier, production is collected And the slow virus for obtaining can inhibit HSV-1 duplication and proliferation is concentrated.The present invention verifies the slow virus pair through In vitro cell model The toxicity of HSV-1 has inhibiting effect, can resist HSV-1 and infect and HSV-1 is inhibited to be proliferated.

Detailed description of the invention

Fig. 1 is that stable expression Cas9 and gRNA cell line RT-PCR provided in an embodiment of the present invention detects Cas9 expression of results (1% Ago-Gel, Gelred dyeing), g1-g3: three difference gRNA；

Fig. 2 is the cellular morphology provided in an embodiment of the present invention infected after corresponding Vero cell with MOI=0.01HSV-1；

Fig. 3 measures drop with TCID50 method to be provided in an embodiment of the present invention with the corresponding Vero cell of wild type HSV-1 infection Degree, DPI indicate number of days after infection.

Fig. 4 is that provided in an embodiment of the present invention infected with fluorescence quantitative PCR method measurement wild type HSV-1 corresponds to Vero cell Afterwards, HSV-1 relative amount in culture medium.

Fig. 5 is that the secondary HSV-1 infection wild type Vero provided in an embodiment of the present invention to correspond to the generation of Vero cell is thin Born of the same parents measure titre.

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.

The present disclosure is not limited to the tools of the construction method or step of recombined lentivirus vector described herein or other compositions Body details.Under disclosed enlightenment, those skilled in the art will be evident that composition disclosed herein and method can be with Different modes is produced, practices, use, implementation and/or formation.

The phraseology and terminology used herein are only used for the purpose of description, are not construed as the limit to the scope of the claims System.As referred in the description and used in claim, the ordinal number of different structure or method steps is indicated, such as the One, second and third, it is not construed as indicating any of any specific structure or step or this structure or step Particular order or configuration.

All methods being described herein can carry out in any suitable order, except herein it is indicated otherwise, or It is obviously contradictory to the context.Any and whole embodiments used or exemplary language (such as " such as ") is provided herein It is intended merely to facilitate disclosure, is not meant to the limitation of any pair of the scope of the present disclosure, Unless Otherwise Requested.Language in specification And the structure shown in attached drawing should not be construed as indicating that the element of any failed call is necessary to theme of the disclosure. The terms "include", "comprise" used herein or " having " and its variant refer to including be listed in element thereafter or its etc. Valence object and additional element.As "include", "comprise" or " having " certain elements are also understood described in embodiment For " being substantially made of those certain elements " and " being made of those certain elements ".

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)).

Unless otherwise noted, unless stated otherwise, following embodiment agents useful for same and material are commercially available.Such as following implementations DMEM high glucose culture medium used in example is purchased from Thermo company.Fetal calf serum is bought from Gibico company.PEI, Polybrene is bought from sigma company.FastDigest Esp3I, T4 ligase, T4PNK and FastAP are bought from Thermo Company.PCR clean up, gel reclaims kit and the small extraction reagent kit of plasmid are purchased from Qiagen company.

In the embodiment of the invention, the embodiment of the invention provides one kind for inhibiting HSV-1 duplication and/or target Mark gRNA, base editing system, method, kit and its application of sequence expression.

Method provided by the present invention for inhibiting HSV-1 duplication and/or target sequence expression, uses the present invention and provides For inhibit HSV-1 replicate and/or target sequence expression gRNA, base editing system or kit, the method includes But it is not limited to the one or more steps of following steps:

One, gRNA is designed:

1) from ncbi database obtain HSV-1 genome sequence (Human herpesvirus 1strain 17, Complete genome.NC_001806.2, GI:820945227), take ICP0 and ICP4 gene DNA sequence；

2) using step 1) acquisition DNA sequence dna as target,

3) http://crispr.mit.edu/ is used

With http://portals.broadinstitute.org/gpp/public/analysis-tools/sgr na- Design gRNA design tool obtains possible gRNA

Sequence takes two kinds of design tool marking highest gRNA sequences.

Designed gRNA sequence are as follows:

ICP0-1:GCCCCCCCTCGAGAGGACGG；(SEQ ID NO.1)

ICP0-2:AGGCGGAGTCGTCGTCATGG；(SEQ ID NO.2)

ICP0-3:ACCAGCTTGGCGTTGCACAG；(SEQ ID NO.3)

ICP4-1:CGGCCCTCATGTTTGACCCG；(SEQ ID NO.4)

ICP4-2:GGGGGTCTTCGCGCGCGTGG；(SEQ ID NO.5)

ICP4-3:GAAGGAGCTGCTGTTGCGCG。(SEQ ID NO.6)

Two, construction recombination plasmid pLV2HSV

(1) connection template synthesis:

ICP0-1:GCCCCCCCTCGAGAGGACGG；

ICP0-2:AGGCGGAGTCGTCGTCATGG；

ICP0-3:ACCAGCTTGGCGTTGCACAG；

ICP4-1:CGGCCCTCATGTTTGACCCG；

ICP4-2:GGGGGTCTTCGCGCGCGTGG；

ICP4-3:GAAGGAGCTGCTGTTGCGCG。

The primer of gRNA as above is directed in Guangzhou Ai Ji biosynthesis:

FP:CACCGNNNNNNNNNNNNNNNNNNNN (SEQ ID NO.8)；

RP:CAAANNNNNNNNNNNNNNNNNNNN (SEQ ID NO.9).

Wherein the N in FP is the sequence of SEQ ID NO.1-6, and the N in RP is the reverse complementary sequence of corresponding sequence.

(2) processing of primer phosphokinase and Gradient annealing: phosphokinase processing is carried out with reference to Thermo PNK scheme, wherein Each 0.5 μM of FP and RP final concentration, Gradient annealing program are as follows: 95 DEG C of 5min then carry out Gradient annealing, outlet temperature with -1 DEG C/s 25℃；Gradient annealing product is connected on the pLentiCRISPR V2 carrier by Esp3I digestion processing, obtains recombinant plasmid pLV2HSV；

Wherein, pLentiCRISPR V2 carrier used is to buy from addgene.

2, the identification of recombinant plasmid pLentiCRISPR V2

To identify pLentiCRISPR V2 recombinant vector correctness, we carry out sequencing mirror to recombinant plasmid pLV2HSV It is fixed.Sequencing result also indicates that annealing template sequence is correctly cloned into pLentiCRISPR V2 carrier.

In pLentiCRISPR V2 carrier, the amino acid sequence of SpCas9 gene order coding is SEQ ID No.7 institute Show:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKN RICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPG EKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTT QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFL KDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKK。

3, Cas9 albumen, the building of gRNA stable expression cell line

(1) by recombination pLentiCRISPR V2 constructed in 1.5 μ g steps 2,1.5 μ g psPAX2,0.5 μ g pMD2.G With 7.5 μ l PEI in 200 μ l opti-mem, it is added in 293T cell culture medium after being placed at room temperature for 20min, 6h after transfection Liquid is changed into fresh culture.The culture medium for collecting 48h after transfecting removes cell fragment with 0.45 μm of syringe filter.Incited somebody to action It filters restrovirus to be added in Vero cell, and adds the polybrene of final concentration of 8 μ g/ml, culture is for 24 hours.Abandon culture containing virus liquid Base adds the fresh complete medium containing 2.5 μ g/ml puromycin, and step sizing 3 days, resisting cell was to stablize table Up to Cas9 albumen, gRNA cell.With Western blot and RT-PCR verifying, it is seen that Cas9 protein expression (Fig. 1).

Fig. 1 is that stable expression Cas9 and gRNA cell line RT-PCR provided in an embodiment of the present invention detects Cas9 expression of results (1% Ago-Gel, Gelred dyeing), g1-g3: three difference gRNA.

4, influence of the detection stable expression cell line to HSV-1 resistance and to HSV-1 proliferation

(1) detection stable expression cell line infects resistance to HSV-1

With TCID50 method gradient dilution virus, a series of wild type HSV-1 of thinner ratios is added to control and expression In the Vero cell of different gRNA, virus titer, the practical titre of control group display virus, experimental group are measured in these cell lines The virus titer of measurement changes, and indicates that cell changes to HSV-1 sensibility.As it can be seen that stablizing the cell pair of expression ICP4-3 Wild type HSV-1 has highest resistance, can resist HSV-1 and infect (Fig. 2, Fig. 3).

Fig. 2 is the cellular morphology provided in an embodiment of the present invention infected after corresponding Vero cell with MOI=0.01HSV-1；

Fig. 3 measures drop with TCID50 method to be provided in an embodiment of the present invention with the corresponding Vero cell of wild type HSV-1 infection Degree, DPI indicate number of days after infection.

(2) influence that detection stable expression cell line is proliferated HSV-1

To probe into the influence that the cell line of expression ICP4-3 is proliferated HSV-1, we are infected with the HSV-1 of MOI=0.001 It compares and experimental group cell 3 days.Since HSV-1 is that fragmentation is grown under in vitro conditions, if virus multiplication, can secondary infection its His cell, and more viruses are discharged, thus more and more viruses can be generated in culture medium supernatant.Pass through quantitative fluorescent PCR Method, we can measure the content of HSV-1 in culture medium supernatant, and which reflects the proliferative capacities of HSV-1.From our reality It tests as it can be seen that the cell for stablizing expression ICP4-3 can obviously reduce the content of HSV-1 in culture medium, thus it has can inhibit HSV- The ability (Fig. 4) of 1 proliferation.

Fig. 4 is that provided in an embodiment of the present invention infected with fluorescence quantitative PCR method measurement wild type HSV-1 corresponds to Vero cell Afterwards, HSV-1 relative amount in culture medium.

(3) secondary HSV-1 is detected to the invasive ability of wild-type cell

Since the Cas9 and gRNA complex formed can cut HSV-1 genome, thus the very possible cutting The invasive ability of HSV-1 can further be influenced.We infect control and experimental group with the wild type HSV-1 of MOI=0.01 respectively Cell collects culture medium supernatant after 2 days.This serial culture is measured respectively with TCID50 method thereafter through wild type Vero cell HSV-1 titre in base supernatant.The index indicates invasive ability of the HSV-1 to wild-type cell of secondary generation.It is tested from us As it can be seen that the ability (Fig. 5) of infection wild-type cell is lost from the secondary HSV-1 that the cell of expression ICP4-3 generates, thus ICP4- 3 can inhibit HSV-1 secondary infection.

Fig. 5 is that the secondary HSV-1 infection wild type Vero provided in an embodiment of the present invention to correspond to the generation of Vero cell is thin Born of the same parents measure titre.

To further illustrate the technology of the present invention effect, SEQ ID NO.1-5 is of the invention according to repeating the step of ICP4-3 Embodiment, effect such as Fig. 2-4.In terms of detecting secondary HSV-1 to the invasive ability of wild-type cell, SEQ ID NO.1- is expressed The ability for the secondary HSV-1 infection wild-type cell that 5 cell generates has by different degrees of inhibition, thus, SEQ ID NO.1-5 can also inhibit HSV-1 secondary infection to a certain extent.

Sequence table

<110>Zhongshan University

<120>for inhibit HSV-1 replicate and/or target sequence expression CRISPR/Cas9 system, method, kit and It is applied

<130> 2017

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213>artificial sequence (artificial synthesized)

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gccccccctc gagaggacgg 20

<210> 2

<211> 20

<212> DNA

<213>artificial sequence (artificial synthesized)

<400> 2

aggcggagtc gtcgtcatgg 20

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (artificial synthesized)

<400> 3

accagcttgg cgttgcacag 20

<210> 4

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<213>artificial sequence (artificial synthesized)

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cggccctcat gtttgacccg 20

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<213>artificial sequence (artificial synthesized)

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gggggtcttc gcgcgcgtgg 20

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<213>artificial sequence (artificial synthesized)

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gaaggagctg ctgttgcgcg 20

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<211> 1384

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<400> 7

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp Lys Arg Pro Ala Ala Thr Lys Lys

1365 1370 1375

Ala Gly Gln Ala Lys Lys Lys Lys

1380

<210> 8

<211> 25

<212> DNA

<213>artificial sequence (artificial synthesized)

<220>

<221> misc_feature

<222> (6)..(25)

<223> n is a, c, g, or t

<400> 8

caccgnnnnn nnnnnnnnnn nnnnn 25

<210> 9

<211> 24

<212> DNA

<213>artificial sequence (artificial synthesized)

<220>

<221> misc_feature

<222> (5)..(24)

<223> n is a, c, g, or t

<400> 9

caaannnnnn nnnnnnnnnn nnnn 24

Claims (10)

1. a kind of gRNA, which is characterized in that the coded sequence of the gRNA includes core shown in SEQ ID NO.1-SEQ ID NO.6 One of nucleotide sequence is a variety of.

2. a kind of for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed, which is characterized in that include Cas9 albumen and gRNA, the coded sequence of the gRNA include in nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.6 It is one or more.

3. as claimed in claim 1 or 2 for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed, One of 1) or 2) it is characterized in that, the gRNA is: 1) including crRNA and tracrRNA, wherein the part of crRNA The partial sequence complementarity of sequence and tracrRNA simultaneously form dimer, wherein crRNA includes boot sequence, the boot sequence Coded sequence include one of nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.6 or a variety of；2) mosaic type list Chain sgRNA, wherein sgRNA is merged by crRNA with tracrRNA, and sgRNA includes boot sequence, the volume of the boot sequence Code sequence includes one of nucleotide sequence shown in SEQ ID NO.1-SEQ ID NO.6 or a variety of.

4. special as claimed in claim 2 for inhibiting the CRISPR/Cas9 system that HSV-1 is replicated and/or target sequence is expressed Sign is, the Cas9 albumen is micrococcus scarlatinae Cas9, staphylococcus aureus Cas9, campylobacter jejuni Cas9, thermophilic Hot streptococcus Cas9, diplococcus meningitidis Cas9, Lachnospira section Cpf1, amino acid coccus Cpf1's is one or more.

5. a kind of non-naturally occurring or engineering composition, which is characterized in that the composition includes one or more loads Body, one or more carriers include component I and component II:

The component I includes the first regulating element, and the coding such as right being operably connected with first regulating element It is required that the coded sequence of Cas9 albumen described in 2；The component II includes the second regulating element, and adjusts member with described second The coded sequence for the coding gRNA as claimed in claim 2 that part is operably connected；Wherein, component I and II be located at it is identical or On different carriers.

6. it is a kind of for inhibiting the method for HSV-1 duplication and/or target sequence expression in the cell of infection HSV-1 virus, It is characterized in that, comprising: premised on to allow to deliver and/or generate a effective amount of gRNA and/or Cas9 albumen in cell, delivering GRNA, CRISPR/Cas9 system as claimed in claim 2 or non-natural as claimed in claim 5 as described in claim 1 Existing or engineering composition, keeps gRNA, CRISPR/Cas9 system and/or composition and cell close and/or connects Touching.

7. a kind of eukaryotic host cell, which is characterized in that include gRNA as described in claim 1, as claimed in claim 2 CRISPR/Cas9 system or as claimed in claim 5 non-naturally occurring or engineering composition.

8. a kind of for inhibiting the kit of HSV-1 duplication and/or target sequence expression in body cell or human body, feature exists In, including gRNA described in claim 1, CRISPR/Cas9 system as claimed in claim 2 or as claimed in claim 5 One of eukaryotic host cell that non-naturally occurring or engineering composition, claim 7 provide is a variety of.

9. a kind of gRNA as described in claim 1, CRISPR/Cas9 system as claimed in claim 2 or such as claim 5 institute The examination of eukaryotic host cell, claim 8 offer that non-naturally occurring or engineering composition, the claim 7 stated provide Application of the agent box in prevention, improvement, and/or treatment HSV-1 related disease.

10. a kind of gRNA as described in claim 1, CRISPR/Cas9 system as claimed in claim 2 or such as claim 5 The eukaryotic host cell that non-naturally occurring or engineering composition, the claim 7 provides is by following one or more Method is applied:

1) the gRNA sequence that claim 1 provides or the CRISPR/Cas9 system that claim 2 provides, claim 5 are provided It is non-naturally occurring or engineering composition, claim 7 provide eukaryotic host cell individually applied；

2) the gRNA sequence that claim 1 provides or the CRISPR/Cas9 system that claim 2 provides, claim 5 are provided Non-naturally occurring or engineering composition, the eukaryotic host cell that provides of claim 7 and operation, biological therapy, exempt from One or more of epidemic disease treatment use in conjunction；

3) the gRNA sequence provided such as claim 1 or claim 2 being provided by the way of delivering in vivo CRISPR/Cas9 system, non-naturally occurring or engineering the composition of claim 5 offer, claim 7 provide true Core host cell is directly delivered to patient's body and is treated；

4) in-vitro transfection technology is first passed through by the gRNA sequence that claim 1 provides or the CRISPR/Cas9 that claim 2 provides Non-naturally occurring or engineering the composition that system, claim 5 provide is mixed with host cell, then will be containing described The non-natural of CRISPR/Cas9 system, claim 5 offer that the gRNA sequence or claim 2 that claim 1 provides provide Defeated time patient's body of host cell of existing or engineering composition implements treatment；

5) the eukaryotic host cell input patient's body provided claim 7 implements treatment.