CN109679989A - A method of improving base editing system editorial efficiency - Google Patents

Abstract

The invention discloses a kind of methods for improving base editing system editorial efficiency.This method comprises the following steps: making expression of receptor sgRNA, hygromycin albumen, Cas9 albumen, cytosine deaminase and ura DNA saccharification enzyme inhibitor, to carry out base editor to the target gene in the acceptor gene group；Receptor is plant callus；During importing plant callus, hygromycin is used to screen resistant calli as selective agent；The concentration of hygromycin is 35-80mg/L.Experiments have shown that, improve the concentration of selective agent hygromycin, SpCas9n can be improved, and (C → T base of D10A) &PmCDA1&UGI base editing system and rAPOBEC1&SpCas9n&UGI base editing system replaces efficiency, and then obtains and efficiently obtain mutant.The present invention has important application value.

Description

A method of improving base editing system editorial efficiency

Technical field

The invention belongs to field of biotechnology, and in particular to a method of improve base editing system editorial efficiency.

Background technique

CRISPR-Cas9 technology has become strong genome edit, be widely applied to many tissues and In cell.CRISPR/Cas9protein-RNA compound is positioned on target spot by guide RNA (guide RNA), and cutting produces The starting DNA repair mechanism of raw DNA double chain fracture (dsDNA break, DSB), then organism meeting instinct repairs DSB.It repairs There are two types of mechanism is general, one is non-homologous end joining (non-homologous end joining, NHEJ), and another kind is Homologous recombination (homology-directed repair, HDR).NHEJ is in the great majority under normal conditions, therefore repairs generation Random indels (insertions or deletions) is more much higher than accurately repairing.Base is accurately replaced, because HDR low efficiency and DNA profiling is needed, so being very restricted using the application that HDR realizes that base is accurately replaced.

2016, two laboratories David Liu and Akihiko Kondo independently report two it is different types of Cytosine base editing machine (cytosine base editor, CBE), principle is directly realized by by using cytidine deaminase Single cytimidine (Cytosine, C) base is edited, and is no longer repaired by generating DSB and starting HDR, is greatly improved C replaces with the base editorial efficiency (i.e. C → T base replacement efficiency) of thymidine (Thymine, T).

PmCDA1(activation-induced cytidine deaminase(AID)ortholog from sea It lamprey) is a kind of cytidine deaminase.In the PmCDA1 editing machine tested, SpCas9n (D10A) &PmCDA1&UGI alkali Base editing system (it contains ura DNA saccharification enzyme inhibitor (uracil DNA glycosylase inhibitor, UGI)) Average mutation rate it is higher, reason has two: first is that UGI can inhibit ura DNA carbohydrase (uracil DNA Glycosylase, UDG) it is catalyzed and removes uracil (Uracil, U) in DNA, second is that SpCas9n (D10A) is produced on non-editor's chain Raw notch, inducible eukaryotic mismatch repair mechanism or long-patch BER (base-excision repair) repair mechanism, promote Make the more Preference reparations of U:G mispairing at U:A.SpCas9n (D10A) is inconjunction with PmCDA1 and navigates to target spot by sgRNA, C on the single stranded DNA of PmCDA1 catalysis non-matching occurs cytimidine deamination reaction and becomes U, by the reparation of DNA so that U and A (adenine, Adenine) pairing matches T and A, to realize the conversion of C to T further through DNA replication dna.

In order to improve working efficiency, job costs are reduced, the raising that C → T base replaces efficiency is always Plant Genome The research direction of base editing system.

Summary of the invention

The purpose of the present invention is improve the editorial efficiency of base editing system.

The present invention protects the method for improving base editing system editorial efficiency first.

The present invention method to be protected for improving base editing system editorial efficiency, concretely method one, it may include such as Lower step: making expression of receptor sgRNA, hygromycin albumen, Cas9 albumen and cytosine deaminase, thus to the acceptor gene Target gene in group carries out base editor.

It is described " to make expression of receptor sgRNA, hygromycin albumen, Cas9 albumen and cytimidine deamination in the above method one Enzyme " can be by by the coding of the encoding gene of the encoding gene of the sgRNA, the hygromycin albumen, the Cas9 albumen Gene and the cytosine deaminase encoding gene import receptor and realize.Receptor is imported specifically, can be placed in a carrier, It can also be placed in different carriers and be directed respectively into receptor.

The present invention method to be protected for improving base editing system editorial efficiency, concretely method two, it may include such as Lower step: make expression of receptor sgRNA, hygromycin albumen, Cas9 albumen, cytosine deaminase and the suppression of ura DNA carbohydrase Preparation, to carry out base editor to the target gene in the acceptor gene group.

It is described " to make expression of receptor sgRNA, hygromycin albumen, Cas9 albumen, cytosine deaminase in the above method two It is saccharified enzyme inhibitor with ura DNA " it can be by by the coding base of the encoding gene of the sgRNA, the hygromycin albumen Cause, the encoding gene of the Cas9 albumen, the cytosine deaminase encoding gene and ura DNA saccharification enzyme inhibitor Encoding gene import receptor realize.Receptor is imported specifically, can be placed in a carrier, different carriers can also be placed on In be directed respectively into receptor.

In any of the above-described method, the receptor can be plant callus.

In any of the above-described method, " importing plant callus " can by infecting, co-culturing, screening step, obtain To edited plant callus.Wherein hygromycin is used to handle plant callus as selective agent in screening step.

In any of the above-described method, during the importing receptor, hygromycin can be used to screen as selective agent Resistant calli.The concentration of the hygromycin is 35-80mg/L (such as 35-50mg/L, 50-80mg/L, 35mg/L, 50mg/L Or 80mg/L).Before the editorial efficiency of base editing system is not up to saturated (i.e. in base editing system editor also C is not converted into T), the concentration of the hygromycin as selective agent is higher, then the editorial efficiency of base editing system is higher.

In any of the above-described method, the hygromycin albumen can be hygromix phosphotransferase.Hygromycin phosphoric acid The amino acid sequence of transferase can by sequence 1 in sequence table from 5 ' ends nucleotide sequence shown in the 10786th to 11811 It translates.Encode the hygromix phosphotransferase gene can as in sequence table sequence 1 from 5 ' ends the 10786th to Shown in 11811.

In any of the above-described method, the Cas9 albumen can be SpCas9n albumen.The amino acid of SpCas9n albumen Sequence can nucleotide sequence shown in the 3010th to 7278 be translated from 5 ' ends by sequence 1 in sequence table.Coding institute The gene for stating SpCas9n albumen can be as shown in sequence 1 the 3010th to 7278 from 5 ' ends in sequence table.

In any of the above-described method, the cytosine deaminase can be PmCDA1 or rAPOBEC1.The ammonia of PmCDA1 Base acid sequence can nucleotide sequence shown in the 7570th to 8193 be translated from 5 ' ends by sequence 1 in sequence table. The amino acid sequence of rAPOBEC1 can by sequence 2 in sequence table from 5 ' ends nucleotide sequence shown in the 3010th to 3696 It translates.The gene for encoding the PmCDA1 can be as shown in sequence 1 the 7570th to 8193 from 5 ' ends in sequence table.It compiles The gene of the code rAPOBEC1 can be as shown in sequence 2 the 3010th to 3696 from 5 ' ends in sequence table.

In any of the above-described method, the amino acid sequence of the ura DNA saccharification enzyme inhibitor can be by sequence table Middle sequence 1 nucleotide sequence shown in the 8215th to 8511 from 5 ' ends is translated.Encode the ura DNA saccharification The gene of enzyme inhibitor can be as shown in sequence 1 the 8215th to 8511 from 5 ' ends in sequence table.

In any of the above-described method, the sgRNA can be according to the target for carrying out base editor expected in acceptor gene group Gene design.

In any of the above-described method, the plant can be a1) or a2) or a3): a1) gramineae plant；A2) rice； A3) rice varieties OryzasativaLcv.Nipponbare.

Any of the above-described the method can be such that the editorial efficiency of base editing system significantly improves.In the volume of base editing system Before collecting efficiency not up to saturation, the concentration of the hygromycin as selective agent is higher, then the editorial efficiency of base editing system is got over It is high.

The present invention also protects X1) or X2).

X1) hygromycin is improving the application in base editing system editorial efficiency.

X2) application of the hygromycin in base editing system editor's acceptor gene group.

Any of the above-described editor can replace for C → T base.

Any of the above-described editorial efficiency can replace efficiency for C → T base.

In one embodiment of the invention, the present inventor constructs carrier S pCas9n&PmCDA1&UGI.It carries SgRNA in body SpCas9n&PmCDA1&UGI is according to three shot designs of table 1.Carrier S pCas9n&PmCDA1&UGI (ring Shape) nucleotide sequence as shown in sequence 1 in sequence table.In sequence 1, from 5 ' to 3 ', the 131st to 467 starts for OsU3 Son nucleotide sequence, the 474th to 550, the 647th to 723 and the 820th to 896 be pre-tRNA nucleotides sequence Column, the 551st to 570 nucleotide sequence for CS650, the 571st to 646, the 744th to 819 and the 917th to 992 For the nucleotide sequence of sgRNA skeleton, the 724th to 743 nucleotide sequence for CS651, the 897th to 916 is CS652's Nucleotide sequence, the 993rd to 1283 nucleotide sequence for OsU3 terminator, the 1290th to 3003 is OsUbq3 promoter Nucleotide sequence, the 3010th to 7278 nucleotide sequence (do not contain terminator codon) for SpCas9n albumen, the 7570th To 8193 nucleotide sequences (not containing terminator codon) for PmCDA1, the 8215th to 8511 nucleotides sequence for UGI Column, the 8518th to 8712 nucleotide sequence for 35S terminator, the 8787th to 10779 nucleosides for ZmUbi1 promoter Acid sequence, the 10786th to 11811 nucleotide sequence for hygromix phosphotransferase, the 11812nd to 12064 whole for Nos Only sub nucleotide sequence.By carrier S pCas9n&PmCDA1&UGI Introduced into Rice kind OryzasativaLcv.Nipponbare (i.e. receptor), with hygromycin As selective agent (hygromycin concentration is 35mg/L, 50mg/L or 80mg/L), kanamycin-resistant callus tissue is obtained；It counts in positive kanamycin-resistant callus tissue C → T base replace efficiency.The result shows that SpCas9n (C of the D10A) &PmCDA1&UGI base editing system to three target spots It is higher that → T base replaces efficiency；With the increase of hygromycin concentration when screening, C → T base of target spot replaces efficiency also therewith Increase, and when the concentration of hygromycin reaches 50mg/L, C → T base replacement efficiency of target spot basically reaches saturation.Also, it sieves With the increase of hygromycin concentration when selecting, kanamycin-resistant callus tissue positive rate is substantially unaffected, holding higher level.

In another embodiment of the present invention, the present inventor constructs carrier rAPOBEC1&SpCas9n& UGI.SgRNA in carrier rAPOBEC1&SpCas9n&UGI is according to the CS650 and CS652 design in table 1.Carrier The nucleotide sequence of rAPOBEC1&SpCas9n&UGI (annular) is as shown in sequence 2 in sequence table.In sequence 2, from 5 ' to 3 ', 131st to 467 nucleotide sequence for OsU3 promoter, the 474th to 550 and the 820th to 896 is pre-tRNA's Nucleotide sequence, the 551st to 570 nucleotide sequence for CS650, the 571st to 646 and the 917th to 992 is The nucleotide sequence of sgRNA skeleton, the 897th to 916 nucleotide sequence for CS652, the 993rd to 1283 terminates for OsU3 The nucleotide sequence of son, the 1290th to 3003 nucleotide sequence for OsUbq3 promoter, the 3010th to 3696 is The nucleotide sequence (not containing terminator codon) of rAPOBEC1, the 3745th to 8013 nucleotide sequence for SpCas9n is (no Contain terminator codon), the 8020th to 8316 nucleotide sequence for UGI, the 8323rd to 8517 core for 35S terminator Nucleotide sequence, the 8592nd to 10584 nucleotide sequence for ZmUbi1 promoter, the 10591st to 11616 is hygromycin phosphorus The nucleotide sequence of sour transferase, the 11643rd to 11858 nucleotide sequence for CaMV35S polyA.By rAPOBEC1& SpCas9n&UGI base editing system Introduced into Rice kind OryzasativaLcv.Nipponbare, using hygromycin as selective agent, (hygromycin concentration is 35mg/L, 50mg/L or 80mg/L), obtain kanamycin-resistant callus tissue；C → T the base counted in positive kanamycin-resistant callus tissue replaces efficiency.As a result Show with SpCas9n (compared with D10A) &PmCDA1&UGI base editing system, rAPOBEC1&SpCas9n&UGI base editor System is lower to C → T base replacement efficiency of CS650 and CS652；But with the increasing of hygromycin concentration in screening and culturing medium Add, C → T base replacement efficiency of target spot is consequently increased；For target spot CS652, when the concentration of hygromycin in screening and culturing medium When reaching 50mg/L, C → T base replacement efficiency of target spot basically reaches saturation.Also, with the increasing of hygromycin concentration when screening Add, kanamycin-resistant callus tissue positive rate keeps higher level.

It is demonstrated experimentally that improving the concentration of the hygromycin as selective agent, SpCas9n (D10A) &PmCDA1& can be improved Editorial efficiency (i.e. C → T base replacement effect of UGI base editing system and rAPOBEC1&SpCas9n&UGI base editing system Rate).It can be seen that improve as selective agent hygromycin concentration, can in the case where not influencing kanamycin-resistant callus tissue positive rate, C → T the base for improving base editing system editor acceptor gene group replaces efficiency, and then efficiently obtains mutant.Present invention tool There is important application value.

Detailed description of the invention

Fig. 1 is that (C → T base of D10A) &PmCDA1&UGI base editing system replaces the experiment knot of efficiency to SpCas9n Fruit.

Fig. 2 is that C → T base of rAPOBEC1&SpCas9n&UGI base editing system replaces the experimental result of efficiency.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.

Three target spots shown in the 2nd column are tested in following 1 selection tables 1 of embodiment, and embodiment 2 selects the 2nd column in table 1 Shown in two target spots tested, target sequence is shown in Table in 1 the 3rd column, and corresponding target gene is shown in Table the 1st column in 1.

Table 1

Target gene	Target spot title	Target sequence
			OsALS	CS650	cgcgtccatggagatccacc
OsCDC48	CS651	gaccagccagcgtctggcgc
			OsNRT1.1B	CS652	cggcgacggcgagcaagtgg

Primer pair 650 is by primer CS650-F:5 '-taagaaccaccagcgacacc-3 ' and primer CS650-R:5 '- Ggtaattgtgcttggtgatggag-3 ' composition, for expanding CS650.

Primer pair 651 is by primer CS651-F:5 '-acatcgagatggagaagcgg-3 ' and primer CS651-R:5 '- Ccatgctccaatcgatgaatac-3 ' composition, for expanding CS651.

Primer pair 652 is by primer CS652-F:5 '-ttacgaactttataactttgtcgg-3 ' and primer CS652-R: 5 '-atggaggcgatgaggaagac-3 ' composition, for expanding CS652.

C → T base replaces efficiency=generation C → T base replacement rice positive kanamycin-resistant callus tissue block number/20 × 100%.

Embodiment 1, high concentration hygromycin selection improve SpCas9n, and (D10A) &PmCDA1&UGI base editing system is to target C → T base of point replaces efficiency

One, the building of gene editing carrier

The present inventor constructs carrier S pCas9n&PmCDA1&UGI.

The nucleotide sequence of carrier S pCas9n&PmCDA1&UGI (annular) is as shown in sequence 1 in sequence table.In sequence 1, From 5 ' to 3 ', the 131st to 467 be OsU3 promoter nucleotide sequence, the 474th to 550, the 647th to 723 and the 820 to 896 be pre-tRNA nucleotide sequence, the 551st to 570 be CS650 nucleotide sequence, the 571st to 646 Position, the 744th to 819 and the 917th to 992 be sgRNA skeleton nucleotide sequence, the 724th to 743 is CS651 Nucleotide sequence, the 897th to 916 nucleotide sequence for CS652, the 993rd to 1283 nucleotide for OsU3 terminator Sequence, the 1290th to 3003 nucleotide sequence for OsUbq3 promoter, the 3010th to 7278 nucleotide for SpCas9n Sequence (does not contain terminator codon), the 7570th to 8193 nucleotide sequence (not containing terminator codon) for PmCDA1, 8215th to 8511 nucleotide sequence for UGI, the 8518th to 8712 nucleotide sequence for 35S terminator, the 8787th It is the nucleotide sequence of ZmUbi1 promoter, the 10786th to 11811 nucleosides for hygromix phosphotransferase to 10779 Acid sequence, the 11812nd to 12064 nucleotide sequence for Nos terminator.

Two, gene editing is carried out in callus

1, carrier S pCas9n&PmCDA1&UGI is imported into Agrobacterium LBA4404 (Shanghai Wei Di Bioisystech Co., Ltd Product, CAT# AC1030), obtain recombinational agrobacterium.

2, recombinational agrobacterium is seeded to the YEP culture medium containing 50 μ g/ml kanamycins and 25 μ g/ml rifampins, 28 DEG C, 150rpm shake culture obtains the culture bacterium solution that OD600nm is 1.0-2.0；Take the culture bacterium solution, room temperature, 10000rpm from Heart 1min collects thallus；The thallus is taken, being resuspended with infected liquid and being diluted to OD600nm is 0.2, obtains Agrobacterium infected liquid.

Infected liquid is that the sugar in N6 fluid nutrient medium is replaced with dextrose and saccharose to obtain；In infected liquid, glucose Concentration be 10g/L, the concentration of sucrose is 20g/L.

3, rice varieties OryzasativaLcv.Nipponbare mature seed decladding threshing is placed in 100mL triangular flask, and 70% (v/v) ethyl alcohol is added Aqueous solution soaking 30sec, then be placed in 25% (v/v) aqueous sodium hypochlorite solution, 120rpm concussion sterilizing 30min, sterile water punching Wash 3 times, with filter paper suck dry moisture, then seed embryo be placed in downward on N6 solid medium, 28 DEG C dark culture 4-6 weeks, obtain Callus.

4, after completing step 3, callus is placed in the Agrobacterium infected liquid containing 100 μM of acetosyringones and impregnates 10min, so First be placed on the culture dish (including about 200mL infected liquid) for being covered with two layers of sterilizing filter paper afterwards, 21 DEG C dark culture 1 day；It then will be more Wound be placed on recovery media (the N6 solid medium containing 200mg/L Ticarcillin/Clavulanate Acid), 25-28 DEG C dark culture 3 days.

5, the callus for taking step 4 to obtain, being placed in screening and culturing medium, (the N6 solid medium of the hygromycin containing 35mg/L contains The N6 solid medium of 50mg/L hygromycin or the N6 solid medium of the hygromycin containing 80mg/L), 28 DEG C dark culture 2 weeks.

6, the callus for taking step 5 to obtain, is again placed in screening and culturing medium, 28 DEG C dark culture 2 weeks, obtain kanamycin-resistant callus tissue.

7, respectively extract 48 pieces of kanamycin-resistant callus tissues genomic DNA and using it as template, using primers F (5 '- Attatgtagcttgtgcgtttcg-3 ') and primer R (5 '-gatgaagagcttatcgacgt-3 ') composition primer pair carry out PCR amplification obtains pcr amplification product；The pcr amplification product is subjected to agarose gel electrophoresis, is then made the following judgment: such as Contain the DNA fragmentation of about 1150bp in fruit pcr amplification product, then corresponding kanamycin-resistant callus tissue is positive kanamycin-resistant callus tissue；If PCR expands Increase production the DNA fragmentation that about 1150bp is not contained in object, then corresponding kanamycin-resistant callus tissue is not positive kanamycin-resistant callus tissue.

Count kanamycin-resistant callus tissue positive rate.Kanamycin-resistant callus tissue positive rate is ratio shared by positive kanamycin-resistant callus tissue in 48 pieces of kanamycin-resistant callus tissues Example.

Kanamycin-resistant callus tissue positive rate statistical result is shown in Table the 2nd column in 2.The result shows that SpCas9n (D10A) &PmCDA1&UGI In base editing system, when hygromycin concentration in screening and culturing medium is between 35-80mg/L, kanamycin-resistant callus tissue positive rate compared with Height reaches 70% or more.

Table 2

Hygromycin concentration in screening and culturing medium	Kanamycin-resistant callus tissue positive rate
		35mg/L	72.1%
50mg/L	87.1%
		80mg/L	70.7%

8, the genomic DNA of the 20 pieces of positive kanamycin-resistant callus tissues obtained respectively using step 7 is as template, using primer pair 650, Primer pair 651 or primer pair 652 carry out PCR amplification, obtain pcr amplification product.

9, the pcr amplification product for obtaining step 8 is sequenced.

Sequencing result is as follows: for CS650, the site that the replacement of C → T base occurs is corresponded to cgTgtccatggagatccacc；For CS651, the site that the replacement of C → T base occurs is corresponded to gaTTagccagcgtctggcgc；For CS652, the site that the replacement of C → T base occurs is corresponded to CggTgacggcgagcaagtgg, Tggcgacggcgagcaagtgg or TggTgacggcgagcaagtgg.

C → T the base for counting CS650, CS651 and CS652 respectively replaces efficiency.Statistical result see Fig. 1 (Hpt-35 be containing The N6 solid medium of 35mg/L hygromycin, Hpt-50 be the hygromycin containing 50mg/L N6 solid medium, Hpt-80 be containing The N6 solid medium of 80mg/L hygromycin).The result shows that (D10A) &PmCDA1&UGI base editing system is to three by SpCas9n C → T base replacement efficiency of a target spot is higher；With the increase of hygromycin concentration in screening and culturing medium, C → T alkali of target spot Base replacement efficiency is consequently increased, and when the concentration of hygromycin in screening and culturing medium reaches 50mg/L, C → T base of target spot Replacement efficiency basically reaches saturation.

Embodiment 2, high concentration hygromycin selection improve rAPOBEC1&SpCas9n&UGI base editing system to the C of target spot → T base replaces efficiency

One, the building of gene editing carrier

The present inventor constructs carrier rAPOBEC1&SpCas9n&UGI.

The nucleotide sequence of carrier rAPOBEC1&SpCas9n&UGI (annular) is as shown in sequence 2 in sequence table.Sequence 2 In, from 5 ' to 3 ', the 131st to 467 nucleotide sequence for OsU3 promoter, the 474th to 550 and the 820th to 896 Be the nucleotide sequence of pre-tRNA, the 551st to 570 be CS650 nucleotide sequence, the 571st to 646 and the 917th Be the nucleotide sequence of sgRNA skeleton to 992, the 897th to 916 be CS652 nucleotide sequence, the 993rd to 1283 Position be OsU3 terminator nucleotide sequence, the 1290th to 3003 be OsUbq3 promoter nucleotide sequence, the 3010th to 3696 nucleotide sequences (not containing terminator codon) for rAPOBEC1, the 3745th to 8013 nucleosides for SpCas9n Acid sequence (does not contain terminator codon), the 8020th to 8316 nucleotide sequence for UGI, and the 8323rd to 8517 is 35S The nucleotide sequence of terminator, the 8592nd to 10584 nucleotide sequence for ZmUbi1 promoter, the 10591st to 11616 For the nucleotide sequence of hygromix phosphotransferase, the 11643rd to 11858 nucleotide sequence for CaMV35S polyA.

Two, gene editing is carried out in rice callus

1, according to the step of 1-7, carrier S pCas9n&PmCDA1&UGI is replaced with carrier in embodiment 1 two RAPOBEC1&SpCas9n&UGI, other steps are constant, obtain positive kanamycin-resistant callus tissue.

Count kanamycin-resistant callus tissue positive rate.Kanamycin-resistant callus tissue positive rate is ratio shared by positive kanamycin-resistant callus tissue in 48 pieces of kanamycin-resistant callus tissues Example.

Kanamycin-resistant callus tissue positive rate statistical result is shown in Table the 2nd column in 3.The result shows that rAPOBEC1&SpCas9n&UGI base In editing system, when hygromycin concentration in screening and culturing medium is between 35-80mg/L, kanamycin-resistant callus tissue positive rate is higher, reaches To 60% or more.

Table 3

Hygromycin concentration in screening and culturing medium	Kanamycin-resistant callus tissue positive rate
		35mg/L	62.8%
50mg/L	68.9%
		80mg/L	75.4%

2, the genomic DNA of the 20 pieces of positive kanamycin-resistant callus tissues obtained respectively using step 1 is as template, using primer pair 650 Or primer pair 652 carries out PCR amplification, obtains pcr amplification product.

3, the pcr amplification product for obtaining step 2 is sequenced.

Sequencing result is as follows: for CS650, the site that the replacement of C → T base occurs is corresponded to

cgTgtccatggagatccacc；For CS652, the site that the replacement of C → T base occurs is corresponded to

CggTgacggcgagcaagtgg, Tggcgacggcgagcaagtgg or TggTgacggcgagcaagtgg.

C → T the base for counting CS650 and CS652 respectively replaces efficiency.Statistical result is shown in that (Hpt-35 is containing 35mg/L to Fig. 2 The N6 solid medium of hygromycin, Hpt-50 are the N6 solid medium of the hygromycin containing 50mg/L, and Hpt-80 is containing 80mg/L tide The N6 solid medium of mycin).The result shows that with SpCas9n (compared with D10A) &PmCDA1&UGI base editing system, RAPOBEC1&SpCas9n&UGI base editing system is lower to C → T base replacement efficiency of CS650 and CS652；But with C → T base replacement efficiency of the increase of hygromycin concentration in screening and culturing medium, target spot is consequently increased；For target spot CS652, When the concentration of hygromycin in screening and culturing medium reaches 50mg/L, C → T base replacement efficiency of target spot basically reaches saturation.

The above results show the increase with hygromycin concentration in screening and culturing medium, and C → T base of target spot replaces efficiency It is consequently increased.

<110>Beijing City Agriculture and Forestry Institute

<120>a kind of method for improving base editing system editorial efficiency

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 18476

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 1

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240

gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480

caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540

ggctggtgca cgcgtccatg gagatccacc gttttagagc tagaaatagc aagttaaaat 600

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt 660

ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 720

gcagaccagc cagcgtctgg cgcgttttag agctagaaat agcaagttaa aataaggcta 780

gtccgttatc aacttgaaaa agtggcaccg agtcggtgca acaaagcacc agtggtctag 840

tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcacggc 900

gacggcgagc aagtgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 960

atcaacttga aaaagtggca ccgagtcggt gctttttttt ttcgttttgc attgagtttt 1020

ctccgtcgca tgtttgcagt tttattttcc gttttgcatt gaaatttctc cgtctcatgt 1080

ttgcagcgtg ttcaaaaagt acgcagctgt atttcactta tttacggcgc cacattttca 1140

tgccgtttgt gccaactatc ccgagctagt gaatacagct tggcttcaca caacactggt 1200

gacccgctga cctgctcgta cctcgtaccg tcgtacggca cagcatttgg aattaaaggg 1260

tgtgatcgat actgcttgct gctaagctta caaattcggg tcaaggcgga agccagcgcg 1320

ccaccccacg tcagcaaata cggaggcgcg gggttgacgg cgtcacccgg tcctaacggc 1380

gaccaacaaa ccagccagaa gaaattacag taaaaaaaaa gtaaattgca ctttgatcca 1440

ccttttatta cctaagtctc aatttggatc acccttaaac ctatcttttc aatttgggcc 1500

gggttgtggt ttggactacc atgaacaact tttcgtcatg tctaacttcc ctttcagcaa 1560

acatatgaac catatataga ggagatcggc cgtatactag agctgatgtg tttaaggtcg 1620

ttgattgcac gagaaaaaaa aatccaaatc gcaacaatag caaatttatc tggttcaaag 1680

tgaaaagata tgtttaaagg tagtccaaag taaaacttat agataataaa atgtggtcca 1740

aagcgtaatt cactcaaaaa aaatcaacga gacgtgtacc aaacggagac aaacggcatc 1800

ttctcgaaat ttcccaaccg ctcgctcgcc cgcctcgtct tcccggaaac cgcggtggtt 1860

tcagcgtggc ggattctcca agcagacgga gacgtcacgg cacgggactc ctcccaccac 1920

ccaaccgcca taaataccag ccccctcatc tcctctcctc gcatcagctc cacccccgaa 1980

aaatttctcc ccaatctcgc gaggctctcg tcgtcgaatc gaatcctctc gcgtcctcaa 2040

ggtacgctgc ttctcctctc ctcgcttcgt ttcgattcga tttcggacgg gtgaggttgt 2100

tttgttgcta gatccgattg gtggttaggg ttgtcgatgt gattatcgtg agatgtttag 2160

gggttgtaga tctgatggtt gtgatttggg cacggttggt tcgataggtg gaatcgtggt 2220

taggttttgg gattggatgt tggttctgat gattgggggg aatttttacg gttagatgaa 2280

ttgttggatg attcgattgg ggaaatcggt gtagatctgt tggggaattg tggaactagt 2340

catgcctgag tgattggtgc gatttgtagc gtgttccatc ttgtaggcct tgttgcgagc 2400

atgttcagat ctactgttcc gctcttgatt gagttattgg tgccatgggt tggtgcaaac 2460

acaggcttta atatgttata tctgttttgt gtttgatgta gatctgtagg gtagttcttc 2520

ttagacatgg ttcaattatg tagcttgtgc gtttcgattt gatttcatat gttcacagat 2580

tagataatga tgaactcttt taattaattg tcaatggtaa ataggaagtc ttgtcgctat 2640

atctgtcata atgatctcat gttactatct gccagtaatt tatgctaaga actatattag 2700

aatatcatgt tacaatctgt agtaatatca tgttacaatc tgtagttcat ctatataatc 2760

tattgtggta atttcttttt actatctgtg tgaagattat tgccactagt tcattctact 2820

tatttctgaa gttcaggata cgtgtgctgt tactacctat ctgaatacat gtgtgatgtg 2880

cctgttacta tctttttgaa tacatgtatg ttctgttgga atatgtttgc tgtttgatcc 2940

gttgttgtgt ccttaatctt gtgctagttc ttaccctatc tgtttggtga ttatttcttg 3000

cagtacgtaa tggactacaa ggaccacgac ggggattaca aagaccacga catagactac 3060

aaggatgacg atgacaaaat ggcaccgaag aaaaaaagga aggtcggaat ccatggcgtt 3120

ccagctgccg ataagaaata ttccatcgga ctcgccattg gcacgaatag cgtcggatgg 3180

gctgttatta ctgatgagta caaagttccg tctaagaagt tcaaggtgct gggcaacaca 3240

gaccgccaca gcataaagaa aaatctcatc ggtgcactcc ttttcgatag tggggagact 3300

gcagaagcga caagattgaa aaggactgcg agaaggcgct atacacggcg taagaataga 3360

atctgctacc ttcaggagat tttctctaac gaaatggcta aggtcgatga cagtttcttt 3420

catagacttg aggaatcgtt cttggttgag gaggataaga aacatgagag gcacccgata 3480

tttggaaaca tcgtggatga ggtcgcatat catgaaaagt accccacaat ctaccacctg 3540

agaaagaaac tcgttgattc caccgacaaa gcggatttga gactcatcta cctcgctctt 3600

gcccatatga taaagttccg cggacacttt ctgatcgagg gcgacctcaa ccctgataat 3660

agcgacgtcg ataagctctt catccagttg gttcaaacct acaatcagct ctttgaggaa 3720

aacccaatta atgctagtgg agtggatgca aaagcgatac tgtcggccag actctccaag 3780

agcagaaggt tggagaacct gatcgctcaa cttcctggag aaaagaaaaa cggtcttttt 3840

gggaatttga ttgccttgtc tctgggcctc acaccaaact tcaagtcaaa ttttgacctc 3900

gctgaggatg ccaaacttca gttgtctaag gatacctatg atgacgatct tgacaatttg 3960

ctggcacaaa ttggcgacca gtacgcggat ctgttcctcg cagcgaagaa tctgagtgat 4020

gctattctcc tttcggacat actcagggtt aacactgaga tcacaaaagc acctttgagt 4080

gcgtcgatga ttaagcgcta tgatgaacat caccaagacc tcactttgct gaaggccctt 4140

gtgcggcagc aattgccaga gaagtacaaa gaaatcttct ttgaccaatc taagaacgga 4200

tacgctggct atattgatgg aggagcttct caggaggaat tctataagtt tatcaaacct 4260

atacttgaga agatggatgg tacagaggaa ctccttgtta aattgaacag agaagatttg 4320

ctgcgcaagc aacggacctt tgacaacgga tcaattccgc atcagataca cctcggcgag 4380

cttcatgcca tccttcgccg gcaggaagat ttctacccct ttttgaagga caaccgcgag 4440

aagatagaaa aaatccttac gttccggatt ccttactatg tgggtccatt ggcaaggggg 4500

aattcccgct ttgcgtggat gactcggaaa agcgaggaaa ctatcacacc gtggaacttc 4560

gaggaagttg tggacaaggg agcttctgcc caatcattca ttgagaggat gactaacttc 4620

gataagaacc tgccgaacga gaaagttctc cccaagcact ccctccttta cgagtatttc 4680

accgtgtata acgaacttac gaaggttaaa tacgtgactg agggtatgag gaagccagca 4740

ttcttgagcg gggaacaaaa gaaagcgatt gttgatttgc tgtttaaaac taatcgcaag 4800

gtgacagtca agcagctcaa agaggattat ttcaagaaaa ttgaatgttt cgactctgtg 4860

gagatatcag gagtcgaaga taggtttaac gcttcccttg gcacatacca tgacctcctt 4920

aagatcatta aggacaaaga tttcctggat aacgaggaaa atgaggacat cctcgaagat 4980

attgttctta ccttgacgct gtttgaggat cgcgaaatga tcgaggaacg gcttaagacg 5040

tatgctcact tgttcgacga taaggttatg aagcagctca agcgtagaag gtacactgga 5100

tggggccgtc tgtctagaaa gctcatcaac ggaatacgtg ataaacaaag tggcaagaca 5160

attttggatt ttctgaagtc ggacggattc gccaacagaa attttatgca gctgattcat 5220

gacgatagtc tcaccttcaa agaggacata cagaaggctc aagtgagtgg tcaaggggat 5280

tcgctgcatg aacacatcgc aaacctcgcg ggttcaccgg ccataaagaa aggaatcctt 5340

caaactgtta aggtcgttga tgagttggtt aaagtgatgg gtaggcacaa gcccgaaaac 5400

atagtgatcg agatggctcg cgaaaatcag actacacaaa aagggcagaa gaactctcgc 5460

gagcggatga aaaggattga ggaaggaatc aaggaactgg gctcacagat tctcaaagag 5520

catccagtcg aaaacacaca gctgcaaaat gagaagctct atctttacta tctccaaaat 5580

ggccgggaca tgtatgttga tcaggagctt gacatcaacc gtttgtccga ctatgatgtg 5640

gaccacattg tcccgcaatc tttccttaag gacgattcaa tcgataataa ggtgttgacc 5700

cggagcgata aaaaccgtgg aaagtctgac aatgtccctt cagaggaagt ggttaagaag 5760

atgaagaact actggagaca attgctgaat gcaaaactga tcacacagag aaagttcgac 5820

aacctcacca aagcagagag aggtgggctc agtgaacttg ataaagcggg cttcattaag 5880

cgtcagctcg ttgagactag acagatcacg aagcatgtcg cgcagatttt ggattcgcgg 5940

atgaacacga agtacgacga gaatgataaa ctgatacgtg aagtcaaggt tatcactctt 6000

aagtccaaat tggtgagcga tttcagaaag gacttccaat tctataaggt cagggagatc 6060

aacaattatc atcacgctca cgatgcctac cttaatgctg ttgtggggac cgcccttatt 6120

aagaaatacc ctaaattgga gtctgaattc gtttacgggg attataaggt ctacgacgtt 6180

aggaaaatga tagctaagag tgagcaggag atcggtaaag caactgcgaa gtatttcttt 6240

tactcgaaca tcatgaattt ctttaagacc gagataacgc tggcaaatgg cgaaattaga 6300

aagaggcctc tcatagagac taacggtgag acaggggaaa tcgtctggga taagggtagg 6360

gactttgcga cagtgcgcaa ggtcctctct atgccgcaag ttaatattgt gaagaaaacc 6420

gaggtgcaga cgggaggctt ctccaaggaa agcatacttc ccaaacggaa ctctgataag 6480

ttgatcgctc gtaagaaaga ttgggaccct aagaaatatg gtgggttcga ttccccaact 6540

gttgcttaca gcgtgctggt cgttgccaag gtcgagaagg gtaaatccaa gaaactcaaa 6600

agcgttaagg aactccttgg gattactatc atggagagat cttcattcga aaagaatcct 6660

atcgactttc ttgaggccaa aggatataag gaagttaaga aagatctgat aatcaaactc 6720

ccaaagtact cattgtttga gctggaaaac ggcaggaagc gcatgcttgc ttccgccgga 6780

gagttgcaga aagggaacga gttggctctg ccttctaagt atgttaactt cctctatctt 6840

gcctctcatt acgagaagct caaaggctca ccagaggaca acgaacagaa acaacttttt 6900

gtcgagcaac ataagcacta tttggatgag attatagaac agatcagtga attctcgaaa 6960

agggttatcc ttgcagatgc gaatcttgac aaggtgttgt ctgcatacaa caaacataga 7020

gataagccga tcagggagca agcggaaaat atcattcacc tcttcactct tacaaacttg 7080

ggtgctcccg ctgccttcaa gtattttgat accacgattg accggaaacg ttacacctca 7140

acgaaggagg tgctggatgc caccctcatc caccaatcta ttaccggact ctacgagact 7200

agaatcgatc tctcacagct cggcggggat aaaagaccag cagcgacgaa aaaggcagga 7260

caggctaaga agaagaaaga gctcggagga ggaggcacgg gaggaggagg ctccgccgag 7320

tatgtgcgcg cgctcttcga cttcaacggc aatgacgagg aggatctccc tttcaagaag 7380

ggcgacatcc tccgcatccg cgataagccg gaggagcagt ggtggaacgc agaggactcc 7440

gagggcaagc ggggcatgat cctggtgcca tacgtcgaga agtacagcgg cgattacaag 7500

gaccacgatg gcgactacaa ggatcatgac atcgattaca aggacgatga cgataagtcc 7560

ggcgtcgaca tgacggacgc ggagtatgtg cgcatccacg agaagctcga tatctacacc 7620

ttcaagaagc agttcttcaa caataagaag tcggtgtccc atcggtgcta cgtcctcttc 7680

gagctgaagc gcaggggaga gcgccgcgcc tgcttctggg gctacgcggt gaataagccg 7740

cagtcaggca cagagcgcgg catccacgcc gagatcttct cgatccggaa ggtcgaggag 7800

tacctccgcg acaacccagg ccagttcacg atcaattggt actccagctg gtccccttgc 7860

gcagattgcg cagagaagat cctcgagtgg tacaaccagg agctgagggg caatggccat 7920

accctcaaga tctgggcctg caagctgtac tacgagaaga acgcgaggaa tcagatcggc 7980

ctctggaacc tgcgggataa tggcgtgggc ctcaacgtga tggtgtccga gcactaccag 8040

tgctgccgca agatcttcat ccagtcctcc cacaatcagc tgaacgagaa taggtggctc 8100

gaaaagaccc tgaagcgcgc cgagaagtgg aggagcgagc tgtctatcat gatccaggtc 8160

aagatcctgc acaccacaaa gtcaccggcg gtgggcggcg gcggcagcga attctccggc 8220

ggcagcacga acctcagcga catcatcgag aaggagacag gcaagcagct cgtgatccag 8280

gagtctatcc tcatgctgcc tgaggaggtg gaggaggtca tcggcaacaa gccggagtcc 8340

gatatcctcg tgcacaccgc ctacgacgag tcgacagatg agaatgtcat gctcctgacc 8400

tccgacgcac cagagtacaa gccatgggcg ctcgtgatcc aggattccaa cggcgagaat 8460

aagatcaaga tgctgtctgg cggctccccg aagaagaagc gcaaggtcta gactagtctg 8520

aaatcaccag tctctctcta caaatctatc tctctctata ataatgtgtg agtagttccc 8580

agataaggga attagggttc ttatagggtt tcgctcatgt gttgagcata taagaaaccc 8640

ttagtatgta tttgtatttg taaaatactt ctatcaataa aatttctaat tcctaaaacc 8700

aaaatccagt ggggcgcccg acctgtactc gcgaaggtta acttacagag agtgtccggg 8760

cgcgcctggt ggatcgtccg cctaggctgc agtgcagcgt gacccggtcg tgcccctctc 8820

tagagataat gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca 8880

cacttgtttg aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa 8940

taatataatc tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag 9000

ttagacatgg tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt 9060

tttagtgtgc atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat 9120

ccattttatt agtacatcca tttagggttt agggttaatg gtttttatag actaattttt 9180

ttagtacatc tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt 9240

agttttttta tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa 9300

acaaataccc tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat 9360

aatgccagcc tgttaaacgc cgtcgacgag tctaacggac accaaccagc gaaccagcag 9420

cgtcgcgtcg ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc 9480

tctcgagagt tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg 9540

gcggagcggc agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcaccgg 9600

cagctacggg ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat 9660

aaatagacac cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac 9720

acacaaccag atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc 9780

gtcctccccc cccccccctc tctaccttct ctagatcggc gttccggtcc atggttaggg 9840

cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc 9900

tgctagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc 9960

cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt 10020

tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata 10080

tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat 10140

gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg 10200

gtggatttat taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg 10260

aagatgatgg atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg 10320

atgcatatac agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg 10380

tcgttcattc gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta 10440

attttggaac tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga 10500

aatatcgatc taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat 10560

ggcatatgca gcatctattc atatgctcta accttgagta cctatctatt ataataaaca 10620

agtatgtttt ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta 10680

tatgtggatt tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt 10740

tgtcgatgct caccctgttg tttggtgtta cttctgcagg agctcatgaa aaagcctgaa 10800

ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg 10860

atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga 10920

tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg 10980

cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga gtttagcgag 11040

agcctgacct attgcatctc ccgccgttca cagggtgtca cgttgcaaga cctgcctgaa 11100

accgaactgc ccgctgttct acaaccggtc gcggaggcta tggatgcgat cgctgcggcc 11160

gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact 11220

acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg 11280

atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc 11340

gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg 11400

acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc 11460

caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag 11520

acgcgctact tcgagcggag gcatccggag cttgcaggat cgccacgact ccgggcgtat 11580

atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat 11640

gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg 11700

cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc 11760

gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaagaaata ggatcgttca 11820

aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 11880

atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 11940

tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 12000

aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 12060

gatctgtagc cctgcaggac gcgtttaatt aagtgcacgc ggccgcctac ttagtcaaga 12120

gcctcgcacg cgactgtcac gcggccagga tcgcctcgtg agcctcgcaa tctgtaccta 12180

gtgtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc 12240

gtttattaga ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg 12300

tatgtgcatg ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc 12360

gctgctatag tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt 12420

cgcacaagtc ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg 12480

tcgcgtgttt tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca 12540

tgaacaagag cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg 12600

acttgaccaa ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga 12660

agatcaccgg caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc 12720

ctggcgacgt tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac 12780

tggacattgc cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt 12840

gggccgacac caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg 12900

agttcgagcg ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc 12960

gaggcgtgaa gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg 13020

agctgatcga ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc 13080

gctcgaccct gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc 13140

ggcgcggtgc cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga 13200

atgaacgcca agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt 13260

cattaccgaa gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc 13320

gcacgtctca accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc 13380

ggcctggccg gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg 13440

tgtatttgag taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa 13500

taaacaaata cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg 13560

tcaggcaaga cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat 13620

gttctgttag tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa 13680

gatcaaccgc taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc 13740

atcggccggc gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg 13800

tccgcgatca aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata 13860

tgggccaccg ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg 13920

ctacaagcgg cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt 13980

gccgaggcgc tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg 14040

agctacccag gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac 14100

gctgcccgcg aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat 14160

gaggtaaaga gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac 14220

gcagcagcaa ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca 14280

actttcagtt gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca 14340

agaccattac cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat 14400

gaataaatga gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa 14460

ccaggcaccg acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta 14520

agcggctggg ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg 14580

gcgtgacggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 14640

ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg 14700

ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc 14760

ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt 14820

cgttccgatg ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt 14880

tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga 14940

cgggcacgta gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct 15000

ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg 15060

agacaagccc ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg 15120

agccgatggc ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac 15180

gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga 15240

gggtgaagcc ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta 15300

catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga 15360

cgtgctgacg gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta 15420

ccgcctggca cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta 15480

cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 15540

cgggtcaaat gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat 15600

cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac 15660

ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc 15720

tgtggatagc acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat 15780

tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga 15840

gaaaaaaggc gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg 15900

cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac 15960

ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 16020

ccgctcaaaa atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc 16080

gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 16140

acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 16200

atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg 16260

cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc 16320

agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 16380

gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 16440

cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 16500

atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 16560

taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 16620

aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 16680

tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 16740

gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 16800

cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 16860

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 16920

atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 16980

tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 17040

ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 17100

acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 17160

aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 17220

aaactcacgt taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa 17280

aatataatat tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc 17340

gacatactgt tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata 17400

ccacttgtcc gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt 17460

cacaaagatg ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt 17520

ttccgtcttt aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca 17580

gttttcgcaa tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg 17640

gctgtctaag ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat 17700

gcactccgca tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga 17760

gcaaaggacg ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa 17820

gtgcaggacc tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg 17880

ttccacatca taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc 17940

attttctccc accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca 18000

gcggtatttt tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta 18060

tttccttcct cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg 18120

aactccaatt cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca 18180

aagttgtttt caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg 18240

tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 18300

atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 18360

gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 18420

ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggct 18476

<210> 2

<211> 18264

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 2

ggtggcagga tatattgtgg tgtaaacatg gcactagcct caccgtcttc gcagacgagg 60

ccgctaagtc gcagctacgc tctcaacggc actgactagg tagtttaaac gtgcacttaa 120

ttaaggtacc gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 180

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 240

gaagccggga acactgggta cgttggaaac cacgtgatgt gaagaagtaa gataaactgt 300

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 360

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 420

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcgga tccaacaaag 480

caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg ttcgattccc 540

ggctggtgca cgcgtccatg gagatccacc gttttagagc tagaaatagc aagttaaaat 600

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt 660

ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt 720

gcagaccagc cagcgtctgg cgcgttttag agctagaaat agcaagttaa aataaggcta 780

gtccgttatc aacttgaaaa agtggcaccg agtcggtgca acaaagcacc agtggtctag 840

tggtagaata gtaccctgcc acggtacaga cccgggttcg attcccggct ggtgcacggc 900

gacggcgagc aagtgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 960

atcaacttga aaaagtggca ccgagtcggt gctttttttt ttcgttttgc attgagtttt 1020

ctccgtcgca tgtttgcagt tttattttcc gttttgcatt gaaatttctc cgtctcatgt 1080

ttgcagcgtg ttcaaaaagt acgcagctgt atttcactta tttacggcgc cacattttca 1140

tgccgtttgt gccaactatc ccgagctagt gaatacagct tggcttcaca caacactggt 1200

gacccgctga cctgctcgta cctcgtaccg tcgtacggca cagcatttgg aattaaaggg 1260

tgtgatcgat actgcttgct gctaagctta caaattcggg tcaaggcgga agccagcgcg 1320

ccaccccacg tcagcaaata cggaggcgcg gggttgacgg cgtcacccgg tcctaacggc 1380

gaccaacaaa ccagccagaa gaaattacag taaaaaaaaa gtaaattgca ctttgatcca 1440

ccttttatta cctaagtctc aatttggatc acccttaaac ctatcttttc aatttgggcc 1500

gggttgtggt ttggactacc atgaacaact tttcgtcatg tctaacttcc ctttcagcaa 1560

acatatgaac catatataga ggagatcggc cgtatactag agctgatgtg tttaaggtcg 1620

ttgattgcac gagaaaaaaa aatccaaatc gcaacaatag caaatttatc tggttcaaag 1680

tgaaaagata tgtttaaagg tagtccaaag taaaacttat agataataaa atgtggtcca 1740

aagcgtaatt cactcaaaaa aaatcaacga gacgtgtacc aaacggagac aaacggcatc 1800

ttctcgaaat ttcccaaccg ctcgctcgcc cgcctcgtct tcccggaaac cgcggtggtt 1860

tcagcgtggc ggattctcca agcagacgga gacgtcacgg cacgggactc ctcccaccac 1920

ccaaccgcca taaataccag ccccctcatc tcctctcctc gcatcagctc cacccccgaa 1980

aaatttctcc ccaatctcgc gaggctctcg tcgtcgaatc gaatcctctc gcgtcctcaa 2040

ggtacgctgc ttctcctctc ctcgcttcgt ttcgattcga tttcggacgg gtgaggttgt 2100

tttgttgcta gatccgattg gtggttaggg ttgtcgatgt gattatcgtg agatgtttag 2160

gggttgtaga tctgatggtt gtgatttggg cacggttggt tcgataggtg gaatcgtggt 2220

taggttttgg gattggatgt tggttctgat gattgggggg aatttttacg gttagatgaa 2280

ttgttggatg attcgattgg ggaaatcggt gtagatctgt tggggaattg tggaactagt 2340

catgcctgag tgattggtgc gatttgtagc gtgttccatc ttgtaggcct tgttgcgagc 2400

atgttcagat ctactgttcc gctcttgatt gagttattgg tgccatgggt tggtgcaaac 2460

acaggcttta atatgttata tctgttttgt gtttgatgta gatctgtagg gtagttcttc 2520

ttagacatgg ttcaattatg tagcttgtgc gtttcgattt gatttcatat gttcacagat 2580

tagataatga tgaactcttt taattaattg tcaatggtaa ataggaagtc ttgtcgctat 2640

atctgtcata atgatctcat gttactatct gccagtaatt tatgctaaga actatattag 2700

aatatcatgt tacaatctgt agtaatatca tgttacaatc tgtagttcat ctatataatc 2760

tattgtggta atttcttttt actatctgtg tgaagattat tgccactagt tcattctact 2820

tatttctgaa gttcaggata cgtgtgctgt tactacctat ctgaatacat gtgtgatgtg 2880

cctgttacta tctttttgaa tacatgtatg ttctgttgga atatgtttgc tgtttgatcc 2940

gttgttgtgt ccttaatctt gtgctagttc ttaccctatc tgtttggtga ttatttcttg 3000

cagtacgtaa tgtccagcga gacaggacca gtggcagtcg acccaacact gcgcaggcgg 3060

atcgagccac acgagttcga ggtgttcttc gatccgaggg agctccggaa ggagacatgc 3120

ctcctgtacg agatcaactg gggcggccgc cactctatct ggaggcatac ctcacagaac 3180

acaaataagc atgtggaggt caacttcatc gagaagttca ccacagagcg gtacttctgc 3240

ccgaatacgc gctgctccat cacctggttc ctgtcgtggt ccccatgcgg agagtgctcg 3300

agggcaatca cggagttcct ctcccgctac ccgcacgtca ccctgttcat ctacatcgca 3360

cggctctacc accatgcgga cccgcggaat aggcagggcc tccgcgatct gatctcttca 3420

ggcgtgacaa tccagatcat gacggagcag gagtcaggct actgctggag gaacttcgtc 3480

aattacagcc catctaacga ggcacactgg ccgcgctacc cgcatctctg ggtgcgcctc 3540

tacgtgctcg agctgtactg catcatcctc ggcctgccgc catgcctcaa tatcctgcgc 3600

aggaagcagc cgcagctgac gttcttcacc atcgccctcc agagctgcca ctaccagcgg 3660

ctccctccgc atatcctgtg ggcgacaggc ctcaagtcag gctcggagac acctggcacg 3720

tccgagagcg ccaccccgga gtctatggac tacaaggacc acgacgggga ttacaaagac 3780

cacgacatag actacaagga tgacgatgac aaaatggcac cgaagaaaaa aaggaaggtc 3840

ggaatccatg gcgttccagc tgccgataag aaatattcca tcggactcgc cattggcacg 3900

aatagcgtcg gatgggctgt tattactgat gagtacaaag ttccgtctaa gaagttcaag 3960

gtgctgggca acacagaccg ccacagcata aagaaaaatc tcatcggtgc actccttttc 4020

gatagtgggg agactgcaga agcgacaaga ttgaaaagga ctgcgagaag gcgctataca 4080

cggcgtaaga atagaatctg ctaccttcag gagattttct ctaacgaaat ggctaaggtc 4140

gatgacagtt tctttcatag acttgaggaa tcgttcttgg ttgaggagga taagaaacat 4200

gagaggcacc cgatatttgg aaacatcgtg gatgaggtcg catatcatga aaagtacccc 4260

acaatctacc acctgagaaa gaaactcgtt gattccaccg acaaagcgga tttgagactc 4320

atctacctcg ctcttgccca tatgataaag ttccgcggac actttctgat cgagggcgac 4380

ctcaaccctg ataatagcga cgtcgataag ctcttcatcc agttggttca aacctacaat 4440

cagctctttg aggaaaaccc aattaatgct agtggagtgg atgcaaaagc gatactgtcg 4500

gccagactct ccaagagcag aaggttggag aacctgatcg ctcaacttcc tggagaaaag 4560

aaaaacggtc tttttgggaa tttgattgcc ttgtctctgg gcctcacacc aaacttcaag 4620

tcaaattttg acctcgctga ggatgccaaa cttcagttgt ctaaggatac ctatgatgac 4680

gatcttgaca atttgctggc acaaattggc gaccagtacg cggatctgtt cctcgcagcg 4740

aagaatctga gtgatgctat tctcctttcg gacatactca gggttaacac tgagatcaca 4800

aaagcacctt tgagtgcgtc gatgattaag cgctatgatg aacatcacca agacctcact 4860

ttgctgaagg cccttgtgcg gcagcaattg ccagagaagt acaaagaaat cttctttgac 4920

caatctaaga acggatacgc tggctatatt gatggaggag cttctcagga ggaattctat 4980

aagtttatca aacctatact tgagaagatg gatggtacag aggaactcct tgttaaattg 5040

aacagagaag atttgctgcg caagcaacgg acctttgaca acggatcaat tccgcatcag 5100

atacacctcg gcgagcttca tgccatcctt cgccggcagg aagatttcta cccctttttg 5160

aaggacaacc gcgagaagat agaaaaaatc cttacgttcc ggattcctta ctatgtgggt 5220

ccattggcaa gggggaattc ccgctttgcg tggatgactc ggaaaagcga ggaaactatc 5280

acaccgtgga acttcgagga agttgtggac aagggagctt ctgcccaatc attcattgag 5340

aggatgacta acttcgataa gaacctgccg aacgagaaag ttctccccaa gcactccctc 5400

ctttacgagt atttcaccgt gtataacgaa cttacgaagg ttaaatacgt gactgagggt 5460

atgaggaagc cagcattctt gagcggggaa caaaagaaag cgattgttga tttgctgttt 5520

aaaactaatc gcaaggtgac agtcaagcag ctcaaagagg attatttcaa gaaaattgaa 5580

tgtttcgact ctgtggagat atcaggagtc gaagataggt ttaacgcttc ccttggcaca 5640

taccatgacc tccttaagat cattaaggac aaagatttcc tggataacga ggaaaatgag 5700

gacatcctcg aagatattgt tcttaccttg acgctgtttg aggatcgcga aatgatcgag 5760

gaacggctta agacgtatgc tcacttgttc gacgataagg ttatgaagca gctcaagcgt 5820

agaaggtaca ctggatgggg ccgtctgtct agaaagctca tcaacggaat acgtgataaa 5880

caaagtggca agacaatttt ggattttctg aagtcggacg gattcgccaa cagaaatttt 5940

atgcagctga ttcatgacga tagtctcacc ttcaaagagg acatacagaa ggctcaagtg 6000

agtggtcaag gggattcgct gcatgaacac atcgcaaacc tcgcgggttc accggccata 6060

aagaaaggaa tccttcaaac tgttaaggtc gttgatgagt tggttaaagt gatgggtagg 6120

cacaagcccg aaaacatagt gatcgagatg gctcgcgaaa atcagactac acaaaaaggg 6180

cagaagaact ctcgcgagcg gatgaaaagg attgaggaag gaatcaagga actgggctca 6240

cagattctca aagagcatcc agtcgaaaac acacagctgc aaaatgagaa gctctatctt 6300

tactatctcc aaaatggccg ggacatgtat gttgatcagg agcttgacat caaccgtttg 6360

tccgactatg atgtggacca cattgtcccg caatctttcc ttaaggacga ttcaatcgat 6420

aataaggtgt tgacccggag cgataaaaac cgtggaaagt ctgacaatgt cccttcagag 6480

gaagtggtta agaagatgaa gaactactgg agacaattgc tgaatgcaaa actgatcaca 6540

cagagaaagt tcgacaacct caccaaagca gagagaggtg ggctcagtga acttgataaa 6600

gcgggcttca ttaagcgtca gctcgttgag actagacaga tcacgaagca tgtcgcgcag 6660

attttggatt cgcggatgaa cacgaagtac gacgagaatg ataaactgat acgtgaagtc 6720

aaggttatca ctcttaagtc caaattggtg agcgatttca gaaaggactt ccaattctat 6780

aaggtcaggg agatcaacaa ttatcatcac gctcacgatg cctaccttaa tgctgttgtg 6840

gggaccgccc ttattaagaa ataccctaaa ttggagtctg aattcgttta cggggattat 6900

aaggtctacg acgttaggaa aatgatagct aagagtgagc aggagatcgg taaagcaact 6960

gcgaagtatt tcttttactc gaacatcatg aatttcttta agaccgagat aacgctggca 7020

aatggcgaaa ttagaaagag gcctctcata gagactaacg gtgagacagg ggaaatcgtc 7080

tgggataagg gtagggactt tgcgacagtg cgcaaggtcc tctctatgcc gcaagttaat 7140

attgtgaaga aaaccgaggt gcagacggga ggcttctcca aggaaagcat acttcccaaa 7200

cggaactctg ataagttgat cgctcgtaag aaagattggg accctaagaa atatggtggg 7260

ttcgattccc caactgttgc ttacagcgtg ctggtcgttg ccaaggtcga gaagggtaaa 7320

tccaagaaac tcaaaagcgt taaggaactc cttgggatta ctatcatgga gagatcttca 7380

ttcgaaaaga atcctatcga ctttcttgag gccaaaggat ataaggaagt taagaaagat 7440

ctgataatca aactcccaaa gtactcattg tttgagctgg aaaacggcag gaagcgcatg 7500

cttgcttccg ccggagagtt gcagaaaggg aacgagttgg ctctgccttc taagtatgtt 7560

aacttcctct atcttgcctc tcattacgag aagctcaaag gctcaccaga ggacaacgaa 7620

cagaaacaac tttttgtcga gcaacataag cactatttgg atgagattat agaacagatc 7680

agtgaattct cgaaaagggt tatccttgca gatgcgaatc ttgacaaggt gttgtctgca 7740

tacaacaaac atagagataa gccgatcagg gagcaagcgg aaaatatcat tcacctcttc 7800

actcttacaa acttgggtgc tcccgctgcc ttcaagtatt ttgataccac gattgaccgg 7860

aaacgttaca cctcaacgaa ggaggtgctg gatgccaccc tcatccacca atctattacc 7920

ggactctacg agactagaat cgatctctca cagctcggcg gggataaaag accagcagcg 7980

acgaaaaagg caggacaggc taagaagaag aaagagctct ccggcggcag cacgaacctc 8040

agcgacatca tcgagaagga gacaggcaag cagctcgtga tccaggagtc tatcctcatg 8100

ctgcctgagg aggtggagga ggtcatcggc aacaagccgg agtccgatat cctcgtgcac 8160

accgcctacg acgagtcgac agatgagaat gtcatgctcc tgacctccga cgcaccagag 8220

tacaagccat gggcgctcgt gatccaggat tccaacggcg agaataagat caagatgctg 8280

tctggcggct ccccgaagaa gaagcgcaag gtctagacta gtctgaaatc accagtctct 8340

ctctacaaat ctatctctct ctataataat gtgtgagtag ttcccagata agggaattag 8400

ggttcttata gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt 8460

atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtggggc 8520

gcccgacctg tactcgcgaa ggttaactta cagagagtgt ccgggcgcgc ctggtggatc 8580

gtccgcctag gctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag ataatgagca 8640

ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg 8700

cagtttatct atctttatac atatatttaa actttactct acgaataata taatctatag 8760

tactacaata atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa 8820

aggacaattg agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg 8880

ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac 8940

atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt 9000

tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa 9060

taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa 9120

gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta 9180

aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca 9240

agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc 9300

tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg 9360

tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct acgggggatt 9420

cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 9480

ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 9540

ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 9600

ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact 9660

tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac 9720

acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 9780

gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 9840

tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 9900

tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 9960

ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 10020

ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 10080

aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 10140

tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 10200

agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 10260

gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 10320

taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 10380

tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 10440

tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 10500

agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 10560

tgttgtttgg tgttacttct gcaggagctc atgaaaaagc ctgaactcac cgcgacgtct 10620

gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag 10680

ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta 10740

aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc 10800

gcgctcccga ttccggaagt gcttgacatt ggggagttta gcgagagcct gacctattgc 10860

atctcccgcc gttcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct 10920

gttctacaac cggtcgcgga ggctatggat gcgatcgctg cggccgatct tagccagacg 10980

agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc 11040

atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc 11100

agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa 11160

gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc 11220

ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc 11280

aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag 11340

cggaggcatc cggagcttgc aggatcgcca cgactccggg cgtatatgct ccgcattggt 11400

cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag 11460

ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc 11520

cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac 11580

cgacgcccca gcactcgtcc gagggcaaag aaatagagta gatgccgacc gggatctgtc 11640

gatcgacaag ctcgagtttc tccataataa tgtgtgagta gttcccagat aagggaatta 11700

gggttcctat agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg 11760

tatttgtaaa atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta 11820

aaatccagat cccccgaatt aattcggcgt taattcagcc tgcaggacgc gtttaattaa 11880

gtgcacgcgg ccgcctactt agtcaagagc ctcgcacgcg actgtcacgc ggccaggatc 11940

gcctcgtgag cctcgcaatc tgtacctagt gtttaaacta tcagtgtttg acaggatata 12000

ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt taaaagggcg 12060

tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttcccctcgg 12120

gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt ctgacgttca 12180

gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg acaggctgcc 12240

gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa gtagaatact 12300

tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg cctgctgggc 12360

tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga actgcacgcg 12420

gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga ccgcccggag 12480

ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac caggctagac 12540

cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca ggaggccggc 12600

gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc cggccgcatg 12660

gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat cgaccgcacc 12720

cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg ccctaccctc 12780

accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg caccgtgaaa 12840

gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact tgagcgcagc 12900

gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga cgcattgacc 12960

gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc atgaaaccgc 13020

accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg gagatgatcg 13080

cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg catgaaatcc 13140

tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc gctgaagaaa 13200

ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg cgtcatgcgg 13260

tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac gcatgaaggt 13320

tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa cccatctagc 13380

ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc cccagggcag 13440

tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg gcatcgaccg 13500

cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag tgatcgacgg 13560

agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact tcgtgctgat 13620

tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg agctggttaa 13680

gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt cgcgggcgat 13740

caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat 13800

tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg ccggcacaac 13860

cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga 13920

aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac 13980

acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg 14040

gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag 14100

ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc 14160

gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa 14220

aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg 14280

tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg 14340

cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc ggcccggtac 14400

aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc 14460

agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc 14520

gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc 14580

ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg 14640

atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg 14700

gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg 14760

gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat 14820

ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg 14880

ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg 14940

tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca 15000

agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg 15060

taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc 15120

gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga 15180

tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag 15240

aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga 15300

agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga 15360

aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg 15420

gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag 15480

gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc 15540

cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt 15600

cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt 15660

taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca 15720

cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg 15780

cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca 15840

atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag 15900

gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 15960

gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 16020

tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga 16080

gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc 16140

ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt 16200

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 16260

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 16320

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 16380

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 16440

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 16500

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 16560

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 16620

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 16680

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 16740

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 16800

gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 16860

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 16920

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 16980

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt 17040

ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct 17100

tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga 17160

tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat 17220

caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt 17280

gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg 17340

gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat 17400

tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg 17460

atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag 17520

ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca 17580

gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt 17640

ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc 17700

tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag 17760

gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg 17820

gtgatattct cattttagcc atttattatt tccttcctct tttctacagt atttaaagat 17880

accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa 17940

ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt 18000

atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta 18060

caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc 18120

gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg 18180

ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc 18240

cggtcgggga gctgttggct ggct 18264

Claims (10)

1. a kind of method for improving base editing system editorial efficiency, includes the following steps: to keep expression of receptor sgRNA, moisture resistance mould Fibroin, Cas9 albumen and cytosine deaminase, to carry out base editor to the target gene in the acceptor gene group.

2. the method as described in claim 1, it is characterised in that: described " to make expression of receptor sgRNA, hygromycin albumen, Cas9 Albumen and cytosine deaminase " is by by the encoding gene of the encoding gene of the sgRNA, the hygromycin albumen, described The encoding gene of Cas9 albumen and the cytosine deaminase encoding gene import receptor and realize.

3. a kind of method for improving base editing system editorial efficiency, includes the following steps: to keep expression of receptor sgRNA, moisture resistance mould Fibroin, Cas9 albumen, cytosine deaminase and ura DNA saccharification enzyme inhibitor, thus in the acceptor gene group Target gene carries out base editor.

4. method as claimed in claim 3, it is characterised in that: described " to make expression of receptor sgRNA, hygromycin albumen, Cas9 Albumen, cytosine deaminase and ura DNA saccharification enzyme inhibitor " is by mould by the encoding gene of the sgRNA, the moisture resistance The encoding gene of fibroin, the encoding gene of the Cas9 albumen, the cytosine deaminase encoding gene and the uracil The encoding gene of DNA saccharification enzyme inhibitor imports receptor and realizes.

5. the method as described in Claims 1-4 is any, it is characterised in that: the receptor is plant callus.

6. method as claimed in claim 1 to 5, it is characterised in that: during the importing receptor, use hygromycin Resistant calli is screened as selective agent.

7. method as claimed in claim 6, it is characterised in that: the concentration of the hygromycin is 35-80mg/L.

8. the method as described in claim 5 to 7 is any, it is characterised in that: the plant be a1) a2) or a3): a1) grass Section plant；A2) rice；A3) rice varieties OryzasativaLcv.Nipponbare.

9.X1) or X2):

X1) hygromycin is improving the application in base editing system editorial efficiency；

X2) application of the hygromycin in base editing system editor's acceptor gene group.

10. method as described in any of the claims 1 to 8 or application as claimed in claim 9, it is characterised in that: the editor Efficiency is that C → T base replaces efficiency.