CN109777832A - MAN2B1 relevant to mannosidosis is repaired in base editor simulationC2248TThe reagent and method of mutation - Google Patents

Abstract

The present invention provides a kind of base editor simulation, repair MAN2B1 relevant to mannosidosis^C2248TThe reagent and method of mutation.Efficient simulation MAN2B1^C2248TThe kit of mutation includes base editing system and for MAN2B1^C2248TThe mutation mt-sgRNA in site；Efficiently repair MAN2B1^C2248TThe kit of mutation includes base editing system and for MAN2B1^C2248TThe mutation re-sgRNA in site.The present invention utilizes base editing technique, by accurately CT or AG single base mutation, so as to simulate, repair MAN2B1^C2248TMutation, thus for treat such be mutated caused by mannosidosis provide the method for highly effective and safe.

Description

MAN2B1 relevant to mannosidosis is repaired in base editor simulationC2248TIt is prominent The reagent and method of change

Technical field

The present invention relates to gene repair fields, more specifically, in people's cell using base editor simulation, repair with it is sweet Dew glucosides stores up the relevant MAN2B1 of disease^C2248TThe reagent and method of (α-mannosidosis) related mutation.

Background technique

α-mannosidosis (α-mannosidosis) is a kind of autosomal recessive inheritance metabolic disease, mainly by It mutates in the gene M AN2B1 of encoding Mannosidase, leads to rare lysosome storage caused by alpha-Mannosidase deficiency Product disease¹.This disease disease incidence is about 1/500000-1/1000000, there is morbidity in the world.Its clinical symptoms is more Sample, there is no a unified, exclusive clinical phenotypes, are mainly shown as immune deficiency, impaired hearing, face distortion, bone Dysplasia, backwardness and other complication².Early stage symptomatic treatment is mainly used to α-mannosidosis at present, is wrapped Include hematopoietic stem cell transplantation, enzyme replacement treatment³.Wherein candidate stem cell treatment must be balanced against the relevant morbidity of entire therapeutic process The risk of rate and the death rate is currently not an optimal selection；Enzyme replacement treatment is temporary relief of symptoms, nor A kind of optimal selection.Therefore specific short there is no thoroughly to eradicate such disease at present.It is mainly in view of α-mannosidosis As caused by MAN2B1 single gene mutation, being repaired to mutated gene will be the effective means for eradicating such disease.

The development of high throughput sequencing technologies and universal, allows us to obtain more mutation relevant to disease, and How repairing to these mutation will be the problem of must facing the genome times afterwards comprehensively.In bacterium or archeobacteria CRISPR/Cas technology has had become that gene editing field is with fastest developing speed, answers since being applied to editor's mammalian cell With most wide technology, the revolution in gene editing field is caused⁴.Currently, CRISPR/Cas9 system has been successfully used to The knockout of DNA is knocked in, is substituted, modifying, marking, the research such as RNA modification and genetic transcription adjusting^5,6.And it is successfully applied to The gene editing of multiple species^6,7.The gene editing that CRISPR/Cas9 is mediated is logical at sgRNA (single guided RNA) Cross target sequence complementation guidance Cas9 albumen position shearing double-stranded DNA, cause double-strand DNA cleavage (double-strand breaks, DSB), under conditions of no template, non-homologous end joining reparation occurs, causes frameshift mutation (frameshift Mutation), lead to gene knockout (knockout)；It under conditions of having template, is repaired by homologous recombination, realizes base Because knocking in (knockin), due to HDR low efficiency (integration seldom occurs), and nonhomologous end engagement mechanisms are easy to produce Radom insertion and deletion (indel), so that new base may be randomly incorporated into, near breaking point so as to cause inaccurate base Because of editor⁸。

Based on base editing technique constructed by CRISPR/Cas9 technology (Base editing), there is cytimidine alkali at present Base editing machine (CBEs, cytosine base editors) and guanine base editing machine (ABEs, Adenine base editors).It can accurately and effectively introduce point mutation in target gene, without double-strand DNA cleavage or any confession Body template shows very big gene editing potentiality^9,10.Currently with base editor, in yeast, plant, mammal and people It is corrected in class cell and genetic diversity Journal of Sex Research^11,12.MAN2B1 gene is located on No. 19 chromosome of the mankind, the gene Overall length 21.5kb¹³.At present HGMD mutation database include and MAN2B1 gene mutation reported in the literature up to more than 100 kinds, In more than half be missense or nonsense mutation.One of missense mutation c.2248C > T occurrence frequency is about 27.5%.This hair It is bright that base editing technique will be utilized to realize safe and efficient simulation and repair MAN2B1 in the cell of people^C2248TMutation^14,15, it is Mannosidosis caused by clinical treatment is accordingly mutated provides reliable reagent and method.

Bibliography

1.Paciotti,S,Codini,M,Tasegian,A,Ceccarini,MR,Cataldi,S,Arcuri,C,et al.(2017).Lysosomal alpha-mannosidase and alpha-mannosidosis.Front Biosci- Landmrk 22:157-167.

2.Lund,AM,Borgwardt,L,Cattaneo,F,Ardigo,D,Geraci,S,Gil-Campos,M,et al.(2018).Comprehensive long-term efficacy and safety of recombinant human alpha-mannosidase(velmanase alfa)treatment in patients with alpha- mannosidosis.Journal of inheritedmetabolic disease.

The molecular genetics of Wu Xiaoyun, Zhang Jie, and Guo Yibin 3. (2013) α-mannosidosis and its in clinic Meaning molecular diagnosis and treatment magazine 5:261-267. in terms of diagnosis and treatment

4.Gaj,T,Gersbach,CA,and Barbas,CF(2013).ZFN,TALEN,and CRISPR/Cas- based methods for genome engineering.Trends in Biotechnology 31:397-405.

5.Hsu,Patrick D,Lander,Eric S,and Zhang,F(2014).Development and Applications of CRISPR-Cas9for Genome Engineering.Cell 157:1262-1278.

6.Komor,AC,Badran,AH,and Liu,DR(2017).CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.Cell 168:20-36.

7.Barrangou,R,and Doudna,JA(2016).Applications of CRISPR technologies in research and beyond.Nature Biotechnology 34:933.

8.Kosicki,M,Tomberg,K,and Bradley,A(2018).Repair of double-strand breaks induced by CRISPR–Cas9leads to large deletions and complex rearrangements.Nature biotechnology.

9.Komor,AC,Kim,YB,Packer,MS,Zuris,JA,and Liu,DR(2016).Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533:420-424.

10.Gaudelli,NM,Komor,AC,Rees,HA,Packer,MS,Badran,AH,Bryson,DI,et al. (2017).Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.Nature 551:464-471.

11.Ren,B,Yan,F,Kuang,Y,Li,N,Zhang,D,Zhou,X,et al.(2018).Improved Base Editor for Efficiently Inducing Genetic Variations in Rice with CRISPR/Cas9- Guided Hyperactive hAID Mutant.Molecular Plant 11:623-626.

12.Nishida,K,Arazoe,T,Yachie,N,Banno,S,Kakimoto,M,Tabata,M,et al. (2016).Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science 353.

13.Hilde Monica Frostad Riise,Thomas Berg, Nilssen,Giovanni Romeo,Ole Kristian Tollersrud,and Ceccherini,I(1997).Genomic structure of the human Lysosomal alpha-Mannosidase Gene(MANB).Genomics.

14.Riise Stensland,HM,Klenow,HB,Van Nguyen,L,Hansen,GM,Malm,D,and Nilssen,O(2012).Identification of 83novel alpha-mannosidosis-associated sequence variants:functional analysis of MAN2B1missense mutations.Human mutation 33:511-520.

15.de Carvalho,TG,da Silveira Matte,U,Giugliani,R,and Baldo,G(2015) .Genome Editing:Potential Treatment for Lysosomal Storage Diseases.Current Stem Cell Reports 1:9-15.

Summary of the invention

The object of the present invention is to provide a kind of efficient simulation, repair MAN2B1^C2248TThe kit and method of mutation.

In order to achieve the above object, the present invention provides a kind of production mannosidosis MAN2B1^C2248TThe side of mutation Method characterized by comprising in people's cell, using CRISPR/Cas9 combination ssODN or utilize the method for base editor Production contains MAN2B1^C2248TCell, this method includes gene editing system and used mt-sgRNA.

When preferably, using CRISPR/Cas9 combination ssODN method, the corresponding DNA sequence dna of mt-sgRNA used is SEQ ID NO.1, ssODN sequence used are SEQ ID NO.2；When using base editor, the corresponding DNA sequence dna of mt-sgRNA used For SEQ ID NO.3.

Preferably, using CRISPR/Cas9 combination ssODN method make be mutated when, Cas9 used be spCas9 or XCas9。

Preferably, using base editing be mutated when, editing system used be EE-XBE3, YE2-XBE3 or YEE-XBE3 preferably selects EE-XBE3.

Preferably, described to contain MAN2B1^C2248TMutant cell behaviour HEK293T cell or people candidate stem cell (HSC)。

Preferably, described to contain MAN2B1^C2248TMutant cell construction method are as follows: according to MAN2B1^C2248TSite Design mutation mt-sgRNA and corresponding mutation ssODN；The expression vector for constructing mt-sgRNA, in vitro by Cas9 albumen and transcription The mode and electricity of mt-sgRNA composition RNP combination ssODN out turns cell, PCR product deep sequencing identification mutation Efficiency, and pick out homozygous mutation cell strain.

Preferably, described to contain MAN2B1^C2248TMutant cell construction method are as follows: according to MAN2B1^C2248TSite Design mutation mt-sgRNA, constructs the expression vector of mt-sgRNA, using base editing system EE-XBE3 or YE2-XBE3 or Person YEE-XBE3 and mutation mt-sgRNA transfect corresponding cell, identify mutation efficiency.

MAN2B1 is efficiently repaired the present invention provides a kind of^C2248TThe kit of mutation, it is characterised in that: containing MAN2B1^C2248TMutant cell in, using be directed to MAN2B1^C2248TThe reparation re-sgRNA guidance base editing system in site arrives Mutational site carries out base editor reparation, and the cell after collecting transfection identifies repair rate.This kit and method include that base is compiled It collects system (base editor) and is directed to MAN2B1^C2248TThe reparation re-sgRNA in site.

Preferably, the base editing system is spCas9-ABE, XCas9-ABE, preferential XCas9-ABE.

Preferably, the base editing system can be plasmid, mRNA or protein form, preferentially select protein form.

Preferably, described to be directed to MAN2B1^C2248TThe sequence of the reparation re-sgRNA in site, corresponding DNA sequence dna are SEQ ID NO.4。

Preferably, used sgRNA can be plasmid form, is also possible to rna form, preferentially selects rna form.

Preferably, the base editor repairs mannosidosis mutation method further include: Sanger sequencing detection Editorial efficiency；The efficiency of high-flux sequence on-target, indel and off-target.

The present invention provides a kind of to make mutation in people's cell and repair the combination of mutation, which is characterized in that according to MAN2B1^C2248TMutation mt-sgRNA and corresponding mutation ssODN are designed for CRISPR/Cas9 method in site；For base Compilation and design is mutated mt-sgRNA；For MAN2B1^C2248TRe-sgRNA and corresponding base editor system are repaired in site design System.

Preferably, the sequence of the mt-sgRNA be the sequence of SEQ ID NO.1, ssODN be SEQ ID NO.2 or The sequence of mt-sgRNA is that the sequence of SEQ ID NO.3, re-sgRNA are SEQ ID NO.4.

The mutation of MAN2B1 is to cause the main reason of mannosidosis, will from this mutation is genetically thoroughly repaired It is the treatment most effective measure of the disease.Base editing system provides a kind of accurate change DNA, i.e., C is become T or A and become For the method for G.

Inventor will be contained using based on the production of the methods of homologous recombination of base editor or CRISPR/Cas9 combination ssODN MAN2B1^C2248TThe cell strain of mutation combines suitable sgRNA to repair this mutation, utilizes depth using base editor thereafter The mode of sequencing detects remediation efficiency and situation of missing the target.And height is provided to treat mannosidosis caused by such is mutated The method for imitating safety.

Detailed description of the invention

Figure 1A to Fig. 1 D is to make MAN2B1 using CRISPR/Cas9 combination ssODN in HEK293T cell line^C2248TIt is prominent The method of change: Figure 1A is the position of corresponding mutation on chromosome.Pathogenic mutation will cause the change of amino acid, become Arg Trp；Figure 1B is can to make the cell strain being accordingly mutated in the way of CRISPR/Cas9 combination ssODN, and ssODN is a length of 113nt, the mutation mt-sgRNA used indicate that black italic is PAM sequence used with underscore；Fig. 1 C is to utilize Cas9 egg White and mt-sgRNA combination ssODN mode electricity turns HEK293T cell, unicellular by airflow classification, to monoclonal cell into Row genotype identification；Fig. 1 D is the cell strain sequencing peak figure of the homozygous mutation obtained.

Fig. 2A -2C is to obtain MAN2B1 using base editing technique^C2248TMutant cell: Fig. 2A is that corresponding mutation is being dyed Position on body.Pathogenic mutation will cause the change of amino acid, and Arg is made to become Trp；Fig. 2 B is to utilize base editor, EE- XBE3/YE2-XBE3/YEE-XBE3 makes corresponding mutant clone, and the mutation mt-sgRNA used is indicated with underscore, black Color italic is PAM sequence used；Fig. 2 C is three kinds of base editing systems to pathogenic sites and its closes on the editorial efficiency in site, C5 is pathogenic sites.

Fig. 3 A-3D is to be repaired using base editing system to mutant clone: Fig. 3 A is to be clicked through using ABE to target position The ideograph that row is repaired, the reparation re-sgRNA used indicate that black italic is PAM sequence used with underscore；Fig. 3 B is Target site is repaired using XABE and ABE system, T6 is corresponding pathogenic sites；Fig. 3 C be to the different form of XABE into The comparison of row remediation efficiency has protein form, mRNA form and plasmid form respectively；Fig. 3 D is to carry out to the cell strain repaired Sequencing, it was demonstrated that the specificity repaired.

Fig. 4 A and Fig. 4 B are that mutant clone carries out analysis of missing the target: Fig. 4 A is to pass through high-flux sequence side to repair cell strain Method detects indel；Fig. 4 B is site progress high-flux sequence detection of missing the target to reparation sgRNA potential 10 used in XABE off-target。

Fig. 5 A and Fig. 5 B are that HSC cell is mutated and is repaired using base editing system: Fig. 5 A is the CD34 obtained⁺ The ratio of positive cell；Fig. 5 B is to be mutated HSC cell using base editor, and the ratio of pathogenic mutation is about 60% (Control), Edited HSC is repaired by base editor, and the ratio for the HSC pathogenic mutation repaired using XABE is about 27% It (XABE), is 55% (ABE) using the ratio of the ABE HSC pathogenic mutation repaired.

Specific embodiment

Clear, complete description is carried out to embodiment of the present invention below in conjunction with embodiment, it is clear that described reality It applies example and is merely to illustrate a part of the embodiments of the present invention, and be not construed as limiting the scope of the invention.It is not specified in embodiment Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer, It is considered as the conventional products that can be obtained by commercially available purchase.

The foregoing is merely present pre-ferred embodiments, are not intended to limit the invention, all in spirit of the invention Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Embodiment 1

In the present embodiment, Cas9/sgRNA combination ssODN production mutation MAN2B1 is utilized on cell strain^C2248TMutation is thin Born of the same parents' strain, this method will be realized using the RNA combination ssODN form of Cas9mRNA and sgRNA, referring to Figure 1A-Fig. 1 D.

1.1 plasmid construction

Near mutational site, design mutation mt-sgRNA (SEQ ID NO.1) synthesizes oligos, upstream sequence are as follows: 5 '-taggCTACACAGACAGCAATGGCC-3 ' (SEQ ID NO. (5)), downstream sequence are as follows: 5 '- AaacGGCCATTGCTGTCTGTGTAG-3 ' (SEQ ID NO. (6)), upstream and downstream sequence by program (95 DEG C, 5min；95℃- 85℃at-2℃/s；85℃-25℃at-0.1℃/s；4 DEG C of hold at) annealing, it is connected to by BsaI (NEB:R0539L) On the PUC57-T7sgRNA carrier of linearisation on (addgene:51132).Linearisation system is as follows: PUC57-T7sgRNA 2μg；6 μ L of buffer (NEB:R0539L)；BsaI 2μL；ddH₂O polishing is to 60 μ L.37 DEG C of digestions are stayed overnight.It is used homologous Template ssODN (SEQ ID NO.4), by PAGE purifying in the way of by Sheng Gong biotech firm (http: // Www.sangon.com/ it) synthesizes.Linked system is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, linearized vector 20ng, Oligo segment (10 μM) 5 μ L, T4 ligase (NEB:M0202L) 0.5 the μ L, ddH of annealing₂L.16, O polishing DEG C was connected to 10 μ Night.The carrier of connection chooses bacterium by conversion, identifies, identifies primer upstream sequence: 5 '-CGCCAGGGTTTTCCCAGTCACGAC- 3 ' (SEQ ID NO.7), downstream sequence are the downstream sequence of corresponding oligo.To positive colony shake bacterium extract plasmid (Axygene: AP-MN-P-250G) measurement concentration is spare.The mutant plasmid of acquisition is named as mt-T7-sgRNA.

The in-vitro transcription of 1.2sgRNA

Using the mt-T7sgRNA of building as template, the segment containing sgRNA, the primer are as follows: F:5 '-are expanded TCTCGCGCGTTTCGGTGATGACGG-3’,(SEQ ID NO.8)R:5’- AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3'(SEQ ID NO.9).Amplification system is as follows: 2Xbuffer (promise is only praised: P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；Template 1ng；Archaeal dna polymerase (promise is only praised: P505)0.5μL；ddH₂O polishing is to 50 μ L.Amplify the PCR product come to purify by following step: every 100 μ L volume adds 4 μ L RNAsecure (Life:AM7005)；60 DEG C 15 minutes；PCR-A (Axygen:AP-PCR-250G) mistake of three times volume is added Column, centrifugation, 12000 revs/min are centrifuged 1 minute；500 μ L W2 are added, are centrifuged 1 minute；Idle running 1 minute；Be added 20 μ L without RNAase water elution.

It is transcribed using in-vitro transcription kit (Ambion, Life Technologies, AM1354), steps are as follows:

Reaction system are as follows: 1 μ L of reaction buffer；enzyme mix 1μL；A 1μL；T 1μL；G1μL；C 1μL； Template 800ng；H₂O polishing is to 10 μ L.37 DEG C of 5 hours of reaction after above-mentioned system mixes.1 μ L DNase, 37 DEG C of reactions are added 15 minutes.Using the sgRNA of QIAquick Gel Extraction Kit (Ambion, Life Technologies, AM1908) recycling transcription, step is such as Under: it above walks reaction volume and 90 μ L Elution solution transplanting 1.5mlEP pipe is added；350 μ L Binding are added Solution is mixed；The mixing of 250 μ L dehydrated alcohols is added；Upper prop；10000 revs/min are centrifuged 30 seconds, outwell waste liquid；It is added 500 μ L Washing solution, 10000 revs/min are centrifuged 30 seconds, outwell waste liquid；Idle running 1 minute；Collecting pipe is changed, 100 μ are added L Elution solution elution；10 μ L ammonium acetates (Ambion, Life Technologies, AM1908) mixing is added；Add Enter the mixing of 275 μ L dehydrated alcohols；- 20 DEG C are placed 30 minutes, while being prepared 70% ethyl alcohol and being placed -20 DEG C；13000 under 4 DEG C of environment Rev/min centrifugation 15 minutes.Supernatant is abandoned, 500 μ L, 70% ethyl alcohol is added；Centrifugation 5 minutes, siphons away waste liquid, dries 5 minutes；It is added The water of 20 μ L dissolves；1 μ L is taken to survey concentration.

The in-vitro transcription of 1.3Cas9

SpCas9 xCas9 digestion recycling.This step is to linearize plasmid Cas9.System is as follows: Cas9 10 μg；10 μ L of buffer I (NEB:R0539L)；4 μ L (NEB:R0539L) of BbsI；H₂O polishing is to 100 μ L.After mixing, 37 DEG C Digestion is stayed overnight.

The recycling of linearization plasmid.4 μ L RNAsecure (Life:AM7005), 60 DEG C of reactions 10 are added in digestion products Minute；It carries out operating remaining step using QIAquick Gel Extraction Kit (QIAGEN:28004), 5 times of volume buffer PB is added, cross column； 750 μ L buffer PE centrifugation is added；Idle running 1 minute；With 10 μ L water elutions, concentration is measured.

It is transcribed in vitro.Sequentially add system according to the requirement of kit (Invitrogen:AM1345): 1, which enters g linearisation, carries Body；10μL2XNTP/ARCA；Polishing is to 20 μ L water；2μL T7ezyme mix；2μL 10xreaction buffer.Mix it It reacts 2 hours for 37 DEG C afterwards.1 μ L DNasea is added to react 15 minutes.

Tailing.Transcription product carries out the stability that tailing processing guarantees transcript mRNA.Specific system is as follows: 20 μ L reaction produces Object；36μL H₂O；20μL 5xE-PAP buffer；10μL 25mM MnCl₂；10μL ATP solution；4μL PEP.Reaction It is reacted 30 minutes for 37 DEG C after system mixes.

Recycling.(QIAGEN:74104) is carried out using QIAquick Gel Extraction Kit.Steps are as follows: above walking reaction product and 350 μ L are added buffer RLT；250 μ L dehydrated alcohols are added, cross column, centrifugation；500 μ L RPE are added, are centrifuged, 500 μ L RPE, centrifugation is added； Idle running；30 μ L water elutions are added.- 80 DEG C of preservations after measurement concentration.

The culture of 1.4 cells and electricity turn

(1) by taking HEK293T cell (being purchased from ATCC) as an example, the present invention carries out the culture and transfection of eukaryotic cells: HEK293T cell inoculation is incubated in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein Containing penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) transfection the first two hour changes the culture medium of antibiotic-free into, using LONZA transfection reagent (SF KIT) according to saying Bright book transfection, cell is by counting to obtain 1X10⁶It is a.By mRNA, sgRNA and the ssODN of Cas9 according to 3 μ g's, 1.5 μ g and 3 μ g Mass mixing.Cell after electric carryover sequence uses DS150, electricity to turn is cultivated two days in the plate of 6cm.

(3) cell sorts unicellular culture by flow sorter, after waiting two weeks, identifies genotype by cracking, splits The ingredient for solving liquid is 50mM KCl, 1.5mM MgCl₂, 10mM Tris pH 8.0,0.5%Nonidet P-40,0.5% Tween20,100μg/ml protease K.The cell strain for selecting homozygous mutation expands culture.

The screening and identification of 1.5 mutant clones

By identifying the monoclonal cell of sorting, in 14 clones of selection, #9 clone is that a homozygote is prominent Become (Fig. 1 D), this cell strain is expanded into culture and carries out subsequent application.

Embodiment 2

In the present embodiment, made on cell strain using base editing system CBEs (Cytosine base editors) It is mutated MAN2B1^C2248TMutant clone, this method will utilize the rna form of the mRNA and sgRNA of corresponding BE3 to realize, referring to Fig. 2A -2C.

1.1 plasmid construction

SgRNA vector construction: near mutational site, design mutation mt-sgRNA (SEQ ID NO.3), synthesis Oligos, upstream sequence are as follows: 5 '-taggTGGCCGGGAGATCCTGGAGA-3 ' (SEQ ID NO. (10)), downstream sequence are as follows: 5 '-aaacTCTCCAGGATCTCCCGGCCA-3 ' (SEQ ID NO. (11)), upstream and downstream sequence by program (95 DEG C, 5min； 95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；4 DEG C of hold at) annealing, be connected to by BsaI (NEB: R0539L) on the PUC57-T7sgRNA carrier linearized on (addgene:51132).Linearisation system is as follows: PUC57- T7sgRNA 2μg；6 μ L of buffer (NEB:R0539L)；BsaI 2μL；ddH₂O polishing is to 60 μ L.37 DEG C of digestions are stayed overnight.Connection System is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, linearized vector 20ng, the 5 μ L of oligo segment (10 μM) of annealing, T4 ligase (NEB:M0202L) 0.5 μ L, ddH₂O polishing to 10 μ, L.16 stay overnight by a DEG C connection.The carrier of connection is chosen by conversion Primer upstream sequence: 5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ' (SEQ ID NO.12), downstream sequence is identified in bacterium, identification It is classified as the downstream sequence of corresponding oligo.Bacterium is shaken to positive colony and extracts plasmid (Axygene:AP-MN-P-250G) measurement concentration It is spare.The mutant plasmid of acquisition is named as mt-T7-sgRNA.

The building of different type BE3: the BE3 of different editions, i.e. EE-XBE3 (SEQ ID NO.13), YE2-XBE3 are constructed (SEQ ID NO.14),YEE-XBE3(SEQ ID NO.15).Original version XBE3 (SEQ ID NO.16) is purchased from Addgene (108380)。

The synthesis of different type APOBEC1: above-mentioned three kinds of BE3 only difference in the encoder block of rAPOBEC1, EE- RAPOBEC1, YE2-rAPOBEC1, YEE-rAPOBEC1, sequence respectively by raw work biology (http: // Www.sangon.com/ it) synthesizes.The both ends of synthesized segment are respectively added with the restriction enzyme site of NotI and SmaI.The segment of synthesis It is cloned on common pmd19t carrier (TAKARA:6013).

The digestion and purifying of carrier: XBE3 and the carrier of above-mentioned three kinds of synthesis pass through NotI (NEB:R0189L), SmaI (NEB:R0141L) digestion, system are as follows: Buffer (NEB:R0189L) 6uL；Plasmid 2ug；NotI 1μL；SmaI 1μL； ddH₂O polishing is to 60 μ L.In 37 DEG C of overnight digestions after sample mixing.Digestion products pass through 1% Ago-Gel, are recycled (Axygen:AP-GX-250G).Wherein XBE3 recycles the skeleton carrier of large fragment, and synthetic vectors recycle the small fragment of APOBEC1 Carrier.Recycling carries out (Axygen:AP-PCR-250G) according to the operation instruction of kit.Segment after the recovery is passed through 2000 detectable concentration of Nanodrop.

Connection, conversion and the plasmid of carrier extract: the skeleton carrier and APOBEC1 segment of recycling are by connecting, connector It is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, skeleton carrier 20ng, APOBEC1 segment 50ng, T4 ligase (NEB: M0202L) 0.5 μ L, ddH₂O polishing is to 10 μ L, and 16 DEG C of connections are overnight.Step of converting is as follows: taking 20 μ L competent cells (TransGen:CD201) it thaws on ice；The connection product of 2 μ L is mixed with competent cell, is placed 20 minutes on ice；42℃ Heat shock 60 seconds；It places 2 minutes on ice, 400 μ L recovery LB culture mediums (MDBio:L001-1kg) of addition, shaking table 30 minutes；Take 70 μ L applies ammonia benzyl plate, and (50 μ g/ml, 37 DEG C of incubators cultivate 14 hours.Monoclonal bacterium is selected, in 4ml LB liquid medium Expand culture, extracts plasmid (Axygene:AP-MN-P-250G) after 14 hours.Steps are as follows: bacterium solution passes through 4000 revs/min Centrifugation 10 minutes, outwells supernatant culture medium；The buffer S1 of 350 μ L is added, thallus is dispelled, is transferred in 2ml centrifuge tube； The buffer S2 of 250 μ L is added, turns upside down 8 times；The buffer S3 of 250 μ L is added, is mixed by inversion 6 times, generates precipitating； 12000 revs/min are centrifuged 10 minutes, and supernatant is taken to cross column；Waste liquid is outwelled in centrifugation 1 minute, and the W1 of 500 μ L is added, and is centrifuged one point Clock outwells waste liquid；The W2 of 750 μ L is added, is centrifuged, outwells supernatant；The W2 of 500 μ L is added, is centrifuged, outwells supernatant；1 point of idle running Clock；The eluent of 50 μ L is added, stands 2 minutes, centrifugation.It obtains plasmid and passes through Concentration Testing, 10 μ L is taken to send sequencing, positive plasmid It is stored in -20 DEG C.

Finally it is built into following four kinds of BE3:

(1) EE-XBE3, SEQ ID NO.13；

(2) YE2-XBE3, SEQ ID NO.14；

(3) YEE-XBE3, SEQ ID NO.15；

The in-vitro transcription of 1.2sgRNA

Using the mt-T7sgRNA of building as template, the segment containing sgRNA, the primer are as follows: F:5 '-are expanded TCTCGCGCGTTTCGGTGATGACGG-3’,(SEQ ID NO.8)R:5’- AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3'(SEQ ID NO.9).Amplification system is as follows: 2Xbuffer (promise is only praised: P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；Template 1ng；Archaeal dna polymerase (promise is only praised: P505)0.5μL；ddH₂O polishing is to 50 μ L.Amplify the PCR product come to purify by following step: every 100 μ L volume adds 4 μ L RNAsecure (Life:AM7005)；60 DEG C 15 minutes；PCR-A (Axygen:AP-PCR-250G) mistake of three times volume is added Column, centrifugation, 12000 revs/min are centrifuged 1 minute；500 μ L W2 are added, are centrifuged 1 minute；Idle running 1 minute；Be added 20 μ L without RNAase water elution.

It is transcribed using in-vitro transcription kit (Ambion, Life Technologies, AM1354), steps are as follows:

Reaction system are as follows: 1 μ L of reaction buffer；enzyme mix 1μL；A 1μL；T 1μL；G1μL；C 1μL； Template 800ng；H₂O polishing is to 10 μ L.37 DEG C of 5 hours of reaction after above-mentioned system mixes.1 μ L DNase, 37 DEG C of reactions are added 15 minutes.Using the sgRNA of QIAquick Gel Extraction Kit (Ambion, Life Technologies, AM1908) recycling transcription, step is such as Under: it above walks reaction volume and 90 μ L Elution solution transplanting 1.5mlEP pipe is added；350 μ L Binding are added Solution is mixed；The mixing of 250 μ L dehydrated alcohols is added；Upper prop；10000 revs/min are centrifuged 30 seconds, outwell waste liquid；It is added 500 μ L Washing solution, 10000 revs/min are centrifuged 30 seconds, outwell waste liquid；Idle running 1 minute；Collecting pipe is changed, 100 μ are added L Elution solution elution；10 μ L ammonium acetates (Ambion, Life Technologies, AM1908) mixing is added；Add Enter the mixing of 275 μ L dehydrated alcohols；- 20 DEG C are placed 30 minutes, while being prepared 70% ethyl alcohol and being placed -20 DEG C；13000 under 4 DEG C of environment Rev/min centrifugation 15 minutes.Supernatant is abandoned, 500 μ L, 70% ethyl alcohol is added；Centrifugation 5 minutes, siphons away waste liquid, dries 5 minutes；It is added The water of 20 μ L dissolves；1 μ L is taken to survey concentration.

The in-vitro transcription of 1.3XBE3

XBE3 digestion recycling.This step is to linearize different types of XBE3.System is as follows: 10 μ g of XBE3； 10 μ L of buffer I (NEB:R0539L)；4 μ L (NEB:R0539L) of BbsI；H₂O polishing is to 100 μ L.After mixing, 37 DEG C of enzymes Cut through night.

The recycling of linearization plasmid.4 μ L RNAsecure (Life:AM7005), 60 DEG C of reactions 10 are added in digestion products Minute；It carries out operating remaining step using QIAquick Gel Extraction Kit (QIAGEN:28004), 5 times of volume buffer PB is added, cross column； 750 μ L buffer PE centrifugation is added；Idle running 1 minute；With 10 μ L water elutions, concentration is measured.

It is transcribed in vitro.Sequentially add system according to the requirement of kit (Invitrogen:AM1345): 1, which enters g linearisation, carries Body；10μL2XNTP/ARCA；Polishing is to 20 μ L water；2μL T7ezyme mix；2μL 10xreaction buffer.Mix it It reacts 2 hours for 37 DEG C afterwards.1 μ L DNasea is added to react 15 minutes.

Tailing.Transcription product carries out the stability that tailing processing guarantees transcript mRNA.Specific system is as follows: 20 μ L reaction produces Object；36μL H₂O；20μL 5xE-PAP buffer；10μL 25mM MnCl₂；10μL ATP solution；4μL PEP.Reaction It is reacted 30 minutes for 37 DEG C after system mixes.

Recycling.(QIAGEN:74104) is carried out using QIAquick Gel Extraction Kit.Steps are as follows: above walking reaction product and 350 μ L are added buffer RLT；250 μ L dehydrated alcohols are added, cross column, centrifugation；500 μ L RPE are added, are centrifuged, 500 μ L RPE, centrifugation is added； Idle running；30 μ L water elutions are added.- 80 DEG C of preservations after measurement concentration.

The culture of 1.4 cells and electricity turn

(1) by taking HEK293T cell (being purchased from ATCC) as an example, the present invention carries out the culture and transfection of eukaryotic cells: HEK293T cell inoculation is incubated in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein Containing penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) transfection the first two hour changes the culture medium of antibiotic-free into, using LONZA transfection reagent (SF KIT) according to saying Bright book transfection, cell is by counting to obtain 1X10⁶It is a.Quality by two kinds of ingredients of mRNA of XBE3 is respectively 3 μ g, 1.5 μ g.Electricity turns Cell after program uses DS150, electricity to turn is cultivated two days in the plate of 6cm.

(3) high-throughput detection mutation efficiency, shown in Fig. 2 C, three kinds of different XBE3 have the editor of pathogenic sites different Efficiency.Wherein EE-XBE3 and YE2-XBE3 is to pathogenic sites C5 editorial efficiency with higher, reaches 50% or more, and YEE- XBE3 is lower to the editorial efficiency of pathogenic sites.Meanwhile nearby there are other C, EE-XBE3 10% volume to C4 for pathogenic sites Efficiency is collected, YE2-XBE3 has about 20% editorial efficiency.Therefore EE-XBE3 has preferable editor's effect to this pathogenic sites Rate, while there is the editorial efficiency of lower non-pathogenic sites.

Embodiment 3

In the present embodiment, homozygous mutant cells strain to acquisition, using base editing system to MAN2B1^C2248TRealization is repaired It is multiple.The present embodiment will combine the reparation repaired sgRNA and carry out mutational site using XABE and ABE plasmid, referring to Fig. 3 A-3D.

1.1 plasmid construction

Near mutational site, re-sgRNA is repaired in design, synthesizes oligos, upstream sequence are as follows: 5 '-accg CTCCCAGCCATTGCTGTCTG-3 ' (SEQ ID NO. (17)), downstream sequence are as follows: 5 '-aaac CAGACAGCAATGGCtGGGAG-3 ' (SEQ ID NO. (18)), upstream and downstream sequence by program (95 DEG C, 5min；95℃-85 ℃at-2℃/s；85℃-25℃at-0.1℃/s；4 DEG C of hold at) annealing, it is connected to by BsaI (NEB:R0539L) line On the PGL3-U6sgRNA carrier of property on SEQ ID NO. (19) (addgene:51133).Linearisation system is as follows: PGL3-U6sgRNA 2μg；6 μ L of buffer (NEB:R0539L)；BsaI 2μL；ddH₂O polishing is to 60 μ L.37 DEG C of digestions are stayed overnight. Linked system is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, linearized vector 20ng, the oligo segment (10 μM) of annealing 5 μ L, T4 ligase (NEB:M0202L) 0.5 μ L, ddH₂O polishing to 10 μ, L.16 stay overnight by a DEG C connection.The carrier of connection is by turning Change, choose bacterium, identify, identifies primer upstream sequence: 5 '-TTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO.20), downstream sequence It is classified as the downstream sequence of corresponding oligo.Bacterium is shaken to positive colony and extracts plasmid (Axygene:AP-MN-P-250G) measurement concentration It is spare.The mutant plasmid of acquisition is named as re-U6-sgRNA.

The culture of 1.3 cells and electricity turn

(1) the homozygous mutant cells strain inoculated and cultured obtained is in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein containing penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) transfection the first two hour changes the culture medium of antibiotic-free into, using LONZA transfection reagent (SF KIT) according to saying Bright book transfection, cell is by counting to obtain 1X10⁶It is a.By plasmid ABE, sgRNA is according to 3 μ g, the mass mixing of 1.5 μ g.Electric carryover Cell after sequence uses DS150, electricity to turn is cultivated two days in the plate of 6cm.

The detection of 1.4 remediation efficiencies

By carrying out high-flux sequence to the cell after transfection, the remediation efficiency of base editor is analyzed.As shown in Figure 3B, The problem of ABE is because of the PAM sequence identified, it is lower to the editorial efficiency of pathogenic sites (T6).XABE can identify NG sequence, institute To pathogenic sites remediation efficiency with higher.XABE is to MAN2B1^C2248TIt is optimal repair system.

Embodiment 4

In the present embodiment, homozygous mutant cells strain to acquisition, using base editing system to MAN2B1^C2248TRealization is repaired It is multiple.The present embodiment will using the mRNA of XABE, protein binding repair sgRNA carry out mutational site reparation, referring to Fig. 3 A-3D with And Fig. 4 A and Fig. 4 B.

1.1 protein purification

The code area of XABE plasmid is building up in carrier pET28a (the excellent precious biology of SEQ ID NO.21, VT1207).It shakes After about shaking bacterium 4 hours (OD=0.8), IPTG1Mm is added in bacterium, 8L LB, Kana, 16 degree are shaken bacterium 48h.Heavy bacterium, centrifugation 5000g, 20min.It is resuspended, all heavy bacterium is resuspended with bufferA, bacterium must be broken up completely, stifled when preventing broken below Fill in instrument.It is broken, bacterium solution is crossed into broken instruments, until solution is limpid, generally at least it is broken twice.Instrument prepares, and needs to clean 3-4 times, high pressure part metals pipe needs ice bath, needs to clean 3-4 times after instrument use.Collect the 10 full cell crackings of μ L Product, subsequent western detection.Pyrolysis product is placed in 50ml centrifuge tube, 80000g, 40min.Supernatant is collected, in repetition Step is stated, until granule foreign removal is clean.0.45um filter filters supernatant, and 10 μ L is taken to detect for subsequent western, prepares Start solid metallic affinity chromatography (Immobilized Metal Affinity Chromatography (IMAC) (cobalt column).Cobalt After column needs to wash one time with ddH2O, with bufferA rinse several times.Protein sample is crossed into cobalt column (this time with two pillars), and is received Collect efflux.It repeats the above steps and takes 10 μ L samples for subsequent western.Decontamination, with 40mL added with 5mM imidazoles BufferA crosses pillar, to remove the lower impurity of affinity.Efflux is collected, and takes 10 μ L samples for subsequent western.

Elution crosses pillar added with the bufferA of 500mM imidazoles with 30ml, displaces destination protein.Collect purpose egg It is white, and take 10 μ L samples for subsequent western.Cobalt column after elution needs to be cleaned with ddH2O, to remove imidazoles, Zhi Houzai It is balanced with bufferA.Western, destination protein about 160KD configure suitable SDS-PAGE glue, 210V electricity according to albumen size Swimming.After electrophoresis, glue is cut off, is placed in Coomassie brilliant blue, micro-wave oven is heated at high temperature 1min.Later, it is cleaned with ddH2O, Microwave stove heating 20min.After being rinsed with water, take pictures.Protein concentration: the destination protein eluted is added to protein concentration column In, 3900rpm, 20min.Albumen after concentration carries out ion-exchange chromatography (Ion exchange chromatography (IEC)), to remove and protein bound nucleic acid.Under principle, that is, high level salt solution of ion-exchange chromatography, this ionic bond will be by It destroys, to release destination protein.The destination protein collected is chromatographed, digestion is carried out after concentrated, to remove His-tag.

1.2 plasmid construction

Near mutational site, re-sgRNA is repaired in design, synthesizes oligos, upstream sequence are as follows: 5 '-tagg CTCCCAGCCATTGCTGTCTG-3 ' (SEQ ID NO. (15)), downstream sequence are as follows: 5 '-aaac CAGACAGCAATGGCtGGGAG-3 ' (SEQ ID NO. (16)), upstream and downstream sequence by program (95 DEG C, 5min；95℃-85 ℃at-2℃/s；85℃-25℃at-0.1℃/s；4 DEG C of hold at) annealing, it is connected to by BsaI (NEB:R0539L) line On the PUC57-T7sgRNA carrier of property on (addgene:51132).Linearisation system is as follows: PUC57-T7sgRNA 2 μg；6 μ L of buffer (NEB:R0539L)；BsaI 2μL；ddH₂O polishing is to 60 μ L.37 DEG C of digestions are stayed overnight.Linked system is as follows: T4 connection buffer (NEB:M0202L) 1 μ L, linearized vector 20ng, 5 μ L, the T4 ligase of oligo segment (10 μM) of annealing (NEB:M0202L) 0.5 μ L, ddH₂O polishing to 10 μ, L.16 stay overnight by a DEG C connection.The carrier of connection chooses bacterium by conversion, identifies, Identify primer upstream sequence: 5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ' (SEQ ID NO.12), downstream sequence is corresponding The downstream sequence of oligo.It is spare that bacterium extraction plasmid (Axygene:AP-MN-P-250G) measurement concentration is shaken to positive colony.It obtains Mutant plasmid be named as re-T7-sgRNA.

The in-vitro transcription of 1.2sgRNA

Using the re-T7sgRNA of building as template, the segment containing sgRNA, the primer are as follows: F:5 '-are expanded TCTCGCGCGTTTCGGTGATGACGG-3’,(SEQ ID NO.8)R:5’- AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3'(SEQ ID NO.9).Amplification system is as follows: 2Xbuffer (promise is only praised: P505)25μL；dNTP 1μL；F(10pmol/μL)2μL；R(10pmol/μL)2μL；Template 1ng；Archaeal dna polymerase (promise is only praised: P505)0.5μL；ddH₂O polishing is to 50 μ L.Amplify the PCR product come to purify by following step: every 100 μ L volume adds 4 μ L RNAsecure (Life:AM7005)；60 DEG C 15 minutes；PCR-A (Axygen:AP-PCR-250G) mistake of three times volume is added Column, centrifugation, 12000 revs/min are centrifuged 1 minute；500 μ L W2 are added, are centrifuged 1 minute；Idle running 1 minute；Be added 20 μ L without RNAase water elution.

It is transcribed using in-vitro transcription kit (Ambion, Life Technologies, AM1354), steps are as follows:

Reaction system are as follows: 1 μ L of reaction buffer；enzyme mix 1μL；A 1μL；T 1μL；G1μL；C 1μL； Template 800ng；H₂O polishing is to 10 μ L.37 DEG C of 5 hours of reaction after above-mentioned system mixes.1 μ L DNase, 37 DEG C of reactions are added 15 minutes.Using the sgRNA of QIAquick Gel Extraction Kit (Ambion, Life Technologies, AM1908) recycling transcription, step is such as Under: it above walks reaction volume and 90 μ L Elution solution transplanting 1.5mlEP pipe is added；350 μ L Binding are added Solution is mixed；The mixing of 250 μ L dehydrated alcohols is added；Upper prop；10000 revs/min are centrifuged 30 seconds, outwell waste liquid；It is added 500 μ L Washing solution, 10000 revs/min are centrifuged 30 seconds, outwell waste liquid；Idle running 1 minute；Collecting pipe is changed, 100 μ are added LElution solution elution；10 μ L ammonium acetates (Ambion, Life Technologies, AM1908) mixing is added；Add Enter the mixing of 275 μ L dehydrated alcohols；- 20 DEG C are placed 30 minutes, while being prepared 70% ethyl alcohol and being placed -20 DEG C；13000 under 4 DEG C of environment Rev/min centrifugation 15 minutes.Supernatant is abandoned, 500 μ L, 70% ethyl alcohol is added；Centrifugation 5 minutes, siphons away waste liquid, dries 5 minutes；It is added The water of 20 μ L dissolves；1 μ L is taken to survey concentration.

The in-vitro transcription of 1.3XABE

XABE digestion recycling.This step is to linearize plasmid XABE.System is as follows: 10 μ g of XABE；buffer I (NEB:R0539L) 10 μ L；4 μ L (NEB:R0539L) of BbsI；H₂O polishing is to 100 μ L.After mixing, 37 DEG C of digestions are stayed overnight.

The recycling of linearization plasmid.4 μ L RNAsecure (Life:AM7005), 60 DEG C of reactions 10 are added in digestion products Minute；It carries out operating remaining step using QIAquick Gel Extraction Kit (QIAGEN:28004), 5 times of volume buffer PB is added, cross column； 750 μ L buffer PE centrifugation is added；Idle running 1 minute；With 10 μ L water elutions, concentration is measured.

It is transcribed in vitro.Sequentially add system according to the requirement of kit (Invitrogen:AM1345): 1, which enters g linearisation, carries Body；10μL2XNTP/ARCA；Polishing is to 20 μ L water；2μL T7ezyme mix；2μL 10xreaction buffer.Mix it It reacts 2 hours for 37 DEG C afterwards.1 μ L DNasea is added to react 15 minutes.

Tailing.Transcription product carries out the stability that tailing processing guarantees transcript mRNA.Specific system is as follows: 20 μ L reaction produces Object；36μL H₂O；20μL 5xE-PAP buffer；10μL 25mM MnCl₂；10μL ATP solution；4μL PEP.Reaction It is reacted 30 minutes for 37 DEG C after system mixes.

Recycling.(QIAGEN:74104) is carried out using QIAquick Gel Extraction Kit.Steps are as follows: above walking reaction product and 350 μ L are added buffer RLT；250 μ L dehydrated alcohols are added, cross column, centrifugation；500 μ L RPE are added, are centrifuged, 500 μ L RPE, centrifugation is added； Idle running；30 μ L water elutions are added.- 80 DEG C of preservations after measurement concentration.

The culture of 1.4 cells and electricity turn

(1) the homozygous mutant cells strain inoculated and cultured obtained is in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein containing penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) transfection the first two hour changes the culture medium of antibiotic-free into, using LONZA transfection reagent (SF KIT) according to saying Bright book transfection, cell is by counting to obtain 1X10⁶It is a.By the mRNA or albumen of XABE, the sgRNA for transcribing out is according to 3 μ g, 1.5 μ The mass mixing of g.Cell after electric carryover sequence uses DS150, electricity to turn is cultivated two days in the plate of 6cm.

(3) cell sorting after a part transfection is unicellular culture, after waiting two weeks, identifies genotype by cracking, The ingredient of lysate is 50mM KCl, 1.5mM MgCl₂, 10mM Tris pH 8.0,0.5%Nonidet P-40,0.5% Tween 20,100μg/ml protease K.The cell strain for selecting homozygous mutation expands culture.Another part, which is used to identify, to be repaired Multiple efficiency.By carrying out high-flux sequence to the cell after transfection, the remediation efficiency of base editor is analyzed.As shown in Figure 3 C, In terms of three kinds of forms such as albumen, RNA and plasmid, bacterium can repair pathogenic sites XABE, and the albumen of XABE is repaired Multiple efficiency highest.Unicellular to what is sorted out, we also obtain the cell strain (Fig. 3 D) of homozygote reparation.

1.6 miss the target detection

Further, we to the reparation of acquisition clone used in sgRNA potentially miss the target the detection in site, pass through height Flux sequencing discovery, no discovery indel, significantly misses the target without discovery in site of potentially missing the target and (schemes in target site 4A and Fig. 4 B).The above results prove safely and efficiently realize that mannosidosis is relevant using base editing system MAN2B1^C2248TThe reparation of mutation.

Embodiment 5

In the present embodiment, we make MAN2B1 first with EE-XBE3 for the HSC cell extracted^C2248TIt is prominent Become, recycles XABE that reparation sgRNA is combined to repair the cell colony of mutation.

The separation and culture of 1.1HSC cell

Sample of bone marrow is given by hospital, and donor's signed is known book.

The about 50mL marrow specimen of acquisition is transferred in 500mL aseptic empty bottle, PBS is added thereto and is diluted to whole body Product is 280ml, is uniformly mixed.15mL lymphocyte separation medium is respectively added into 8 50mL centrifuge tubes with pipette.Use pipette The marrow specimen that every centrifuge tube is careful thereto, is slowly added into after 35mL dilution.400g is centrifuged 30 minutes.It is sucked out with sputum aspirator At first layer liquid to slightly above buffy coat, it is put into new 50mL centrifuge tube with pipette absorption second layer liquid and mends training Base is supported to 50mL.300g is centrifuged 10 minutes, removes supernatant.Cell is resuspended with 5mL0.5%BSA PBS, then mends to 50mL.It cracks red Cell, is added human red blood cells lysate 1x H-Lyse buffer 10mL, and concussion, pressure-vaccum mixing are stored at room temperature 10min.Add 1x H-wash buffer 10mL is mixed by inversion.Add 0.5%BSA PBS to 50mL, 300g to be centrifuged 10 minutes, abandons supernatant.For 1x10⁸0.3mL 0.5%BSA PBS is added in lymphocyte precipitate cell, and pressure-vaccum is resuspended, then is separately added into 0.1mLFcr Blocking Ab and 0.1mLCD34-beads.It is placed in 4 DEG C of refrigerators 30 minutes, 20mL0.5%BSA PBS is added, be resuspended mixed It is even.300g is centrifuged 10 minutes, removes supernatant.Be added 0.5%BSA PBS to final volume be 4mL.Pillar is put on the top of the shelf and is added 0.5mL 0.5%BSA PBS soaks pillar, and centrifuge tube is placed below pillar for collecting liquid under stream, is stopped to liquid 0.5mL cell suspension is added after drippage into pillar.0.5mL 0.5%BSA PBS cleaning is added after dripping off into pillar by column The cell of son absorption, is repeated 5 times.Pillar is removed from shelf and 1mL 0.5%BSA PBS is added, is pushed away liquid with piston Into 50mL centrifuge tube, it is repeated 3 times.0.5%BSA PBS is added into centrifuge tube to 20mL volume, is added on cell counter, The cell number that first pass crosses pillar is counted under microscope.300g is centrifuged 10 minutes.According to count results, by 1x10⁷Cell is resuspended In 4mL 0.5%BSA PBS, 1 pillar calculating is crossed, is resuspended into the 0.5%BSA PBS of proper volume and excessively an appropriate number of Pillar.CD34+ cell proportion is improved, sorting step is repeated.Count second time cell number for crossing pillar.According to count results, take 5x10⁵CD34+ cell, 0.5%BSA PBS are diluted to 100 μ L (experimental group), take the 100 μ l (control group) of waste liquid for flowing through pillar, CD34, CD3 antibody respectively add 3 μ l, and 4 DEG C put half an hour, and 200 μ LPBS are washed 2 times, and 3500rpm is centrifuged 3min.It is resuspended in 200 μ L PBS carries out flow cytometer detection, as a result, it has been found that the purity that we obtain is up to 97% CD34+ cell, referring to Fig. 5 A.

1.2 protein purification

By EE-XBE3, the code area of XABE and ABE plasmid be building up to carrier pET28a (the excellent precious biology of SEQ ID NO.9, VT1207 in).Bacterium is shaken, after about shaking bacterium 4 hours (OD=0.8), IPTG1Mm is added in 8L LB, Kana, 16 degree are shaken bacterium 48h. Heavy bacterium, is centrifuged 5000g, 20min.It is resuspended, all heavy bacterium is resuspended with bufferA, bacterium must be broken up completely, prevent broken below When block instrument.It is broken, bacterium solution is crossed into broken instruments, until solution is limpid, generally at least it is broken twice.Instrument prepares, It needs to clean 3-4 times, high pressure part metals pipe needs ice bath, needs to clean 3-4 times after instrument use.It is complete to collect 10 μ L Product of cell lysis, subsequent western detection.Pyrolysis product is placed in 50ml centrifuge tube, 80000g, 40min.In collection Clearly, it repeats the above steps, until granule foreign removal is clean.0.45um filter filters supernatant, takes 10 μ L for subsequent western Detection prepares to start solid metallic affinity chromatography (Immobilized Metal Affinity Chromatography (IMAC) (cobalt column).After cobalt column needs to wash one time with ddH2O, with bufferA rinse several times.Protein sample is crossed into cobalt column (this time with two Pillar), and collect efflux.It repeats the above steps and takes 10 μ L samples for subsequent western.Decontamination is added with 40mL There is the bufferA of 5mM imidazoles to cross pillar, to remove the lower impurity of affinity.Efflux is collected, and after taking 10 μ L samples to be used for Continuous western.

Elution crosses pillar added with the bufferA of 500mM imidazoles with 30ml, displaces destination protein.Collect purpose egg It is white, and take 10 μ L samples for subsequent western.Cobalt column after elution needs to be cleaned with ddH2O, to remove imidazoles, Zhi Houzai It is balanced with bufferA.Western, destination protein about 160KD configure suitable SDS-PAGE glue, 210V electricity according to albumen size Swimming.After electrophoresis, glue is cut off, is placed in Coomassie brilliant blue, micro-wave oven is heated at high temperature 1min.Later, it is cleaned with ddH2O, Microwave stove heating 20min.After being rinsed with water, take pictures.Protein concentration: the destination protein eluted is added to protein concentration column In, 3900rpm, 20min.Albumen after concentration carries out ion-exchange chromatography (Ion exchange chromatography (IEC)), to remove and protein bound nucleic acid.Under principle, that is, high level salt solution of ion-exchange chromatography, this ionic bond will be by It destroys, to release destination protein.The destination protein collected is chromatographed, digestion is carried out after concentrated, to remove His-tag.

The culture and transfection of 1.3 candidate stem cells

Isolated CD34+ candidate stem cell culture is added as follows in StemSpan SFEM culture medium, in culture medium Cell factor: the Flt3ligand and small of the Tpo of the SCF of 100ng/mL, 100ng/mL, 100ng/mL Molecules, 1 μM of Stem-Regenin 1, and 0.5 μM of MU729.In 37 DEG C of incubators, 5%CO2 condition is pre- to pierce for cell culture Swash 24 hours.The albumen of corresponding EE-XBE3 is incubated for 10 minutes with corresponding sgRNA at 37 DEG C, LONZA 4D is utilized Neclefector carries out electricity and turns, procedure selection EO-100.Cell after electricity turns continues culture 2 days.

The reparation of 1.4 mutation HSC cell colonys

By detecting to the HSC cell of mutation, discovery has about 60% cell to be mutated in pathogenic sites, Referring to Fig. 5 B.Then, we utilize XABE, ABE and corresponding reparation sgRNA, form RNP electricity in vitro and turn the prominent of above-mentioned acquisition Become cell colony.Cell culture two days later, detects remediation efficiency, finds the HSC cell colony after XABE is repaired Mutation efficiency talked about 28% or so, and for ABE, because of the limitation of its used PAM sequence, mutation efficiency only drops To 55% or so (Fig. 5 B).It is above-mentioned it is experimentally confirmed that in the primary cell of people, it is prominent to causing a disease that we can use base editor Change is repaired, and the patient of mannosidosis caused by this research will accordingly be mutated for clinical treatment provides potentially Therapeutic scheme.

Sequence table

<110>national health State Family Planning Commission Institute Of Science And Technology

<120>reagent and method of base editor simulation, reparation MAN2B1C2248T mutation relevant to mannosidosis

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 1

ctacacagac agcaatggcc ggg 23

<210> 2

<211> 113

<212> DNA

<213>artificial sequence ()

<400> 2

ccgttttgac acaccgctgg agacaaaggg acgcttctac acagacagca atggctggga 60

gatcctggag aggaggtggg gggtgactga gagcactgag ggggtggtct gtg 113

<210> 3

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<212> DNA

<213>artificial sequence ()

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tggccgggag atcctggaga gga 23

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<213>artificial sequence ()

<400> 4

acacagacag caatggccgg gag 23

<210> 5

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<212> DNA

<213>artificial sequence ()

<400> 5

taggctacac agacagcaat ggcc 24

<210> 6

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<212> DNA

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<400> 6

aaacggccat tgctgtctgt gtag 24

<210> 7

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<212> DNA

<213>artificial sequence ()

<400> 7

cgccagggtt ttcccagtca cgac 24

<210> 8

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<212> DNA

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<400> 8

tctcgcgcgt ttcggtgatg acgg 24

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<213>artificial sequence ()

<400> 9

aaaaaaagca ccgactcggt gccacttttt c 31

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taggtggccg ggagatcctg gaga 24

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<213>artificial sequence ()

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aaactctcca ggatctcccg gcca 24

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<212> DNA

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cgccagggtt ttcccagtca cgac 24

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<211> 8532

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atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420

actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480

gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540

gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600

aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660

acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720

tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780

cccgagaatc gacaaggcct ggaagatttg atctcttcag gtgtgactat ccaaattatg 840

actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900

gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960

atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020

ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080

gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140

agtgacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 1200

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 1260

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 1320

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 1380

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 1440

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 1500

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 1560

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 1620

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 1680

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 1740

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 1800

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 1860

cttatcgccc tgtccctcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 1920

gataccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 1980

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 2040

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 2100

atgatcaagc tctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 2160

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 2220

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 2280

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 2340

aaacagcgca ctttcgacaa tggaatcatc ccccaccaga ttcacctggg cgaactgcac 2400

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 2460

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 2520

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgagaaa 2580

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 2640

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 2700

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 2760

tctggagatc agaagaaagc tattgtggac ctcctcttca agacgaaccg gaaagttacc 2820

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 2880

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 2940

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 3000

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 3060

catctcttcg acgacaaagt catgaagcag ctcaagaggc gccgatatac aggatggggg 3120

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 3180

gattttctta agtccgatgg atttgccaac cggaacttca ttcagttgat ccatgatgac 3240

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 3300

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 3360

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 3420

atcgagatgg cccgagagaa ccaaaccacc cagaagggac agaagaacag tagggaaagg 3480

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 3540

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 3600

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 3660

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 3720

gataaaaaca gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 3780

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 3840

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggtttcat caaaaggcag 3900

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 3960

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 4020

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 4080

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 4140

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 4200

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 4260

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 4320

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 4380

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 4440

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 4500

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 4560

tacagtgtac tggttgtggc taaagtggag aaagggaagt ctaaaaaact caaaagcgtc 4620

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 4680

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 4740

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgtgctg 4800

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 4860

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 4920

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 4980

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 5040

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 5100

cctgcagcct tcaagtactt cgacactacc atagacagaa agcggtacac ctctacaaag 5160

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 5220

gacctctctc agctcggtgg agactctggt ggttctacta atctgtcaga tattattgaa 5280

aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340

gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400

agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460

ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520

aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820

ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880

aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940

tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040

acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100

gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160

agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220

agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520

atcaagtgta tc 8532

<210> 14

<211> 8532

<212> DNA

<213>artificial sequence ()

<400> 14

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420

actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480

gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540

gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600

aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660

acctggtttc tcagctatag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720

tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780

ccccgcaatc gacaaggcct ggaagatttg atctcttcag gtgtgactat ccaaattatg 840

actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900

gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960

atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020

ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080

gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140

agtgacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 1200

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 1260

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 1320

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 1380

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 1440

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 1500

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 1560

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 1620

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 1680

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 1740

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 1800

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 1860

cttatcgccc tgtccctcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 1920

gataccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 1980

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 2040

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 2100

atgatcaagc tctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 2160

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 2220

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 2280

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 2340

aaacagcgca ctttcgacaa tggaatcatc ccccaccaga ttcacctggg cgaactgcac 2400

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 2460

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 2520

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgagaaa 2580

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 2640

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 2700

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 2760

tctggagatc agaagaaagc tattgtggac ctcctcttca agacgaaccg gaaagttacc 2820

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 2880

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 2940

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 3000

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 3060

catctcttcg acgacaaagt catgaagcag ctcaagaggc gccgatatac aggatggggg 3120

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 3180

gattttctta agtccgatgg atttgccaac cggaacttca ttcagttgat ccatgatgac 3240

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 3300

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 3360

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 3420

atcgagatgg cccgagagaa ccaaaccacc cagaagggac agaagaacag tagggaaagg 3480

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 3540

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 3600

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 3660

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 3720

gataaaaaca gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 3780

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 3840

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggtttcat caaaaggcag 3900

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 3960

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 4020

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 4080

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 4140

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 4200

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 4260

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 4320

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 4380

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 4440

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 4500

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 4560

tacagtgtac tggttgtggc taaagtggag aaagggaagt ctaaaaaact caaaagcgtc 4620

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 4680

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 4740

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgtgctg 4800

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 4860

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 4920

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 4980

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 5040

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 5100

cctgcagcct tcaagtactt cgacactacc atagacagaa agcggtacac ctctacaaag 5160

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 5220

gacctctctc agctcggtgg agactctggt ggttctacta atctgtcaga tattattgaa 5280

aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340

gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400

agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460

ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520

aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820

ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880

aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940

tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040

acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100

gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160

agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220

agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520

atcaagtgta tc 8532

<210> 15

<211> 8532

<212> DNA

<213>artificial sequence ()

<400> 15

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420

actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480

gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540

gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600

aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660

acctggtttc tcagctacag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720

tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780

cccgagaatc gacaaggcct ggaggatttg atctcttcag gtgtgactat ccaaattatg 840

actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900

gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960

atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020

ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080

gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140

agtgacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 1200

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 1260

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 1320

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 1380

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 1440

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 1500

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 1560

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 1620

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 1680

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 1740

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 1800

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 1860

cttatcgccc tgtccctcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 1920

gataccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 1980

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 2040

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 2100

atgatcaagc tctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 2160

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 2220

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 2280

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 2340

aaacagcgca ctttcgacaa tggaatcatc ccccaccaga ttcacctggg cgaactgcac 2400

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 2460

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 2520

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgagaaa 2580

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 2640

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 2700

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 2760

tctggagatc agaagaaagc tattgtggac ctcctcttca agacgaaccg gaaagttacc 2820

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 2880

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 2940

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 3000

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 3060

catctcttcg acgacaaagt catgaagcag ctcaagaggc gccgatatac aggatggggg 3120

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 3180

gattttctta agtccgatgg atttgccaac cggaacttca ttcagttgat ccatgatgac 3240

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 3300

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 3360

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 3420

atcgagatgg cccgagagaa ccaaaccacc cagaagggac agaagaacag tagggaaagg 3480

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 3540

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 3600

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 3660

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 3720

gataaaaaca gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 3780

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 3840

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggtttcat caaaaggcag 3900

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 3960

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 4020

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 4080

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 4140

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 4200

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 4260

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 4320

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 4380

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 4440

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 4500

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 4560

tacagtgtac tggttgtggc taaagtggag aaagggaagt ctaaaaaact caaaagcgtc 4620

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 4680

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 4740

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgtgctg 4800

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 4860

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 4920

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 4980

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 5040

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 5100

cctgcagcct tcaagtactt cgacactacc atagacagaa agcggtacac ctctacaaag 5160

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 5220

gacctctctc agctcggtgg agactctggt ggttctacta atctgtcaga tattattgaa 5280

aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340

gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400

agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460

ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520

aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820

ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880

aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940

tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040

acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100

gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160

agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220

agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520

atcaagtgta tc 8532

<210> 16

<211> 8532

<212> DNA

<213>artificial sequence ()

<400> 16

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120

ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180

cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240

atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300

ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360

agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420

actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480

gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540

gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600

aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660

acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720

tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780

ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840

actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900

gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960

atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020

ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080

gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140

agtgacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 1200

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 1260

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 1320

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 1380

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 1440

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 1500

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 1560

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 1620

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 1680

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 1740

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 1800

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 1860

cttatcgccc tgtccctcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 1920

gataccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 1980

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 2040

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 2100

atgatcaagc tctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 2160

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 2220

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 2280

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 2340

aaacagcgca ctttcgacaa tggaatcatc ccccaccaga ttcacctggg cgaactgcac 2400

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 2460

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 2520

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgagaaa 2580

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 2640

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 2700

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 2760

tctggagatc agaagaaagc tattgtggac ctcctcttca agacgaaccg gaaagttacc 2820

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 2880

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 2940

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 3000

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 3060

catctcttcg acgacaaagt catgaagcag ctcaagaggc gccgatatac aggatggggg 3120

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 3180

gattttctta agtccgatgg atttgccaac cggaacttca ttcagttgat ccatgatgac 3240

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 3300

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 3360

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 3420

atcgagatgg cccgagagaa ccaaaccacc cagaagggac agaagaacag tagggaaagg 3480

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 3540

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 3600

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 3660

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 3720

gataaaaaca gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 3780

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 3840

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggtttcat caaaaggcag 3900

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 3960

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 4020

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 4080

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 4140

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 4200

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 4260

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 4320

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 4380

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 4440

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 4500

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 4560

tacagtgtac tggttgtggc taaagtggag aaagggaagt ctaaaaaact caaaagcgtc 4620

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 4680

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 4740

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgtgctg 4800

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 4860

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 4920

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 4980

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 5040

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 5100

cctgcagcct tcaagtactt cgacactacc atagacagaa agcggtacac ctctacaaag 5160

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 5220

gacctctctc agctcggtgg agactctggt ggttctacta atctgtcaga tattattgaa 5280

aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340

gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400

agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460

ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520

aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580

gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640

cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700

catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760

agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820

ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880

aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940

tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120

cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180

aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240

aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300

tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360

caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420

cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480

ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540

gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600

agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660

gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720

acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780

gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040

acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100

gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160

agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220

agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520

atcaagtgta tc 8532

<210> 17

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 17

accgctccca gccattgctg tctg 24

<210> 18

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 18

aaaccagaca gcaatggctg ggag 24

<210> 19

<211> 4951

<212> DNA

<213>artificial sequence ()

<400> 19

ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60

cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300

tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata 360

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 420

tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa 480

aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca 540

tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca 600

gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa 660

ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc 720

ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc 780

gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt 840

tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc 900

ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc 960

aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag 1020

gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc 1080

aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct 1140

ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc 1200

acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 1260

gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 1320

ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 1380

gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 1440

ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 1500

ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 1560

cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 1620

gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac 1680

gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 1740

tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa 1800

aggagcgcac gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta 1860

gatcttagcc actttctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata 1920

cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 1980

aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 2040

caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 2100

caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga 2160

gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac 2220

ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct 2280

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 2340

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 2400

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 2460

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 2520

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 2580

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 2640

ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 2700

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 2760

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 2820

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 2880

ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 2940

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 3000

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 3060

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 3120

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 3180

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 3240

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 3300

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca 3360

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 3420

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 3480

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 3540

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 3600

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 3660

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 3720

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 3780

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 3840

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 3900

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 3960

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 4020

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 4080

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 4140

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 4200

cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4260

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 4320

gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 4380

tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 4440

gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 4500

agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 4560

ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 4620

tttaacgcga attttaacaa aatattaacg tttacaattt cccattcgcc attcaggctg 4680

cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta 4740

ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat 4800

ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg 4860

ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa 4920

gtgcaggtgc cagaacattt ctctatcgat a 4951

<210> 20

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 20

tttcccatga ttccttcata 20

<210> 21

<211> 5369

<212> DNA

<213>artificial sequence ()

<400> 21

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata 240

tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggct gctgcccatg 300

gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca 360

caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc 420

ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc 480

gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc 540

gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca 600

ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg 660

caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt 720

tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc 780

agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt 840

ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc 900

ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct 960

gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat 1020

taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg 1080

cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat 1140

cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt 1200

tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca 1260

tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc 1320

gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa 1380

atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat 1440

gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct 1500

ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg 1560

cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat 1620

cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg 1680

cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact 1740

ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 1800

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 1860

acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga 1920

gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac 1980

ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca 2040

ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat 2100

tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg 2160

gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc 2220

tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac 2280

cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa 2340

catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct 2400

gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta 2460

catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca 2520

tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag 2580

taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa 2640

atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc 2700

gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg 2760

aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc 2820

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 2880

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 2940

ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg 3000

gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa 3060

ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 3120

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 3180

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 3240

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 3300

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 3360

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 3420

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 3480

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 3540

cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 3600

aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 3660

gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 3720

agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 3780

ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3840

cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3900

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact 3960

gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc 4020

ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc 4080

tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc 4140

gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat 4200

ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg 4260

tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt 4320

attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg 4380

ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct 4440

cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga 4500

gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc 4560

accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg 4620

gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct 4680

tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca 4740

aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga 4800

gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac 4860

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata 4920

ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 4980

aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 5040

cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 5100

ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 5160

cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 5220

ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 5280

gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 5340

cgccgctaca gggcgcgtcc cattcgcca 5369

Claims (7)

1. a kind of efficient simulation repairs MAN2B1^C2248TThe kit of mutation, which is characterized in that including constructing MAN2B1^C2248TIt is prominent The base editing system BE and corresponding mt-sgRNA of change and be directed to MAN2B1^C2248TThe base editing system ABE of mutation repair And corresponding re-sgRNA.

2. efficient simulation as described in claim 1 repairs MAN2B1^C2248TThe kit of mutation, which is characterized in that described For MAN2B1^C2248TThe sequence of the mutation mt-sgRNA in site is SEQ ID NO.1 or SEQ ID NO.3, for the mutation position The sequence of the reparation re-sgRNA of point is SEQ ID NO.4.

3. efficient simulation as described in claim 1 repairs MAN2B1^C2248TThe kit of mutation, which is characterized in that described Base editing system is EE-XBE3 and XABE.

4. a kind of combination for making mutation and repairing mutation, which is characterized in that including according to MAN2B1^C2248TSite design mutation Mt-sgRNA and corresponding mutation ssODN, it is directed to MAN2B1^C2248TIn the reparation re-sgRNA and base editing system in site At least one.

5. a kind of method that base editor repairs mutation characterized by comprising containing MAN2B1^C2248TMutant cell In, using for MAN2B1^C2248TThe reparation re-sgRNA guidance base editing system in site carries out base editor to mutational site It repairs, the cell after collecting transfection.

6. the method that base editor as claimed in claim 5 repairs mutation, which is characterized in that described contains MAN2B1^C2248T Mutant cell be HEK293T cell or people's HSC cell.

7. the method that base editor as claimed in claim 5 repairs mutation, which is characterized in that described is directed to MAN2B1^C2248T The reparation re-sgRNA in site passes through according to MAN2B1^C2248TRe-sgRNA is repaired in site design, and constructs U6 starting and/or T7 The expression vector of starting obtains.