CN110923229B - CRISPR/Cas9 system for knocking out dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco and application - Google Patents

Abstract

The invention discloses a CRISPR/Cas9 system for knocking out a dmrt1 gene from double gRNA sites in pelteobagrus fulvidraco, wherein the CRISPR/Cas9 system comprises the following steps: (1) the target site 1 is designed on the first exon of the pelteobagrus fulvidraco dmrt1 gene, and the target site 2 is designed on the third exon; (2) designing a primer to detect the accuracy of the target site in the parent fish according to the target site sequence in step (1), amplifying the target site 1 and the nearby sequence by using dmrt 1E 1F and dmrt 1E 1R, and amplifying the target site 2 and the nearby sequence by using dmrt 1E 3F and dmrt 1E 3R; (3) using pUC19-gRNA-scaffold plasmid as template, performing PCR amplification of gRNA1 fragment with dmrt 1E 1gRNA F and gRNA R, and performing PCR amplification of gRNA2 fragment with dmrt 1E 3 gRNA F and gRNA R; taking the PCR product as a template, and carrying out in-vitro transcription and purification to obtain gRNA; (4) in vitro transcription synthesis of Cas9mRNA by using pXT7-hCas9 linearized plasmid as a template; (5) micro-injecting Cas9mRNA and two gRNAs into a pseudobagrus fulvidraco one-cell-stage embryo; (6) and detecting the mutation type and calculating the gene editing rate.

Description

CRISPR/Cas9 system for knocking out dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco and application

Technical Field

The invention relates to the fields of biotechnology and genetic breeding, in particular to a CRISPR/Cas9 system for knocking out a dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco and application thereof.

Background

Pelteobagrus fulvidraco (Pelteobagrus fulvidraco) belongs to Osteichthyes, Clariales, Pseudobagriaceae and Pelteobagrus, and is one of important freshwater economic breeding fishes in China. The growth speed of the male fish of the pelteobagrus fulvidraco is 2-3 times that of the female fish. In recent years, the cultivation of full-orpiment catfishes is promoted in China, but with the rapid development of the cultivation industry of the catfishes, some problems occur in succession, for example, the supply of YY super-male fishes for producing full-male seedlings is short and the price is high; in addition, due to the serious close breeding phenomenon, the germplasm of the pelteobagrus fulvidraco is degraded, the growth speed is reduced, the disease resistance and stress resistance are reduced, and the like. The sustainable development of the yellow catfish breeding industry is severely restricted, so that the existing germplasm of the yellow catfish needs to be purified and rejuvenated urgently, and pure super-male fish and full-female fish with excellent genetic traits are bred.

Understanding the sex determination and differentiation of pelteobagrus fulvidraco and the reproductive regulation mechanism are the basis for artificial sex control breeding. The Dmrt (ble-sex and Mab-3 related transcription factor) gene family contains the consensus sequences of Drosophila's sex-determining gene Dsx and nematode sex-determining gene Mab-3, which encode proteins with zinc finger DNA binding motifs (DM domains) that are an ancient and conserved component of vertebrate sex-determining pathways. At least 8 members of the Dmrt gene family are currently found in a number of species, mammals, birds, fish, reptiles, and amphibians, and Dmrt1 is the most and representative gene among them that has been studied, and plays an important role in the differentiation and development of sex.

The gene editing technology is an important technical means in the field of life science, and the gene function research, disease research and treatment, drug development and the like are accelerated by editing the genome sequence of a specific target gene. Currently, commonly used gene editing technologies mainly include a Zinc-finger nuclease (ZFN) technology, a transcription activator-like effector nuclease (TALEN) technology, and a regularly clustered short interspersed palindromic repeats (CRISPR) system nuclease technology. The first two techniques rely on the DNA binding protein module for DNA sequence recognition, and are relatively complex, time-consuming and expensive in design. The CRISPR technology is used for mediating the recognition of DNA through guide RNA (gRNA), has simple design, high efficiency and low cost, and is widely applied to multiple species.

The full length 18952bp of the pelteobagrus fulvidraco dmrt1 gene comprises 5 exons and 4 introns, and how to select a proper target point makes the whole gene nonfunctional and a phenotype easy to screen appear is the key for successfully obtaining the mutant. The traditional single target spot knockout is low in targeting efficiency, nonsense mutation is easily caused due to high uncertainty of DNA self-repair, deletion of a plurality of bases is not easy to identify, and a large amount of funds are often spent on sequencing identification.

Disclosure of Invention

In view of the above disadvantages, one of the purposes of the present invention is to provide a CRISPR/Cas9 system for knocking out the dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco, so as to obtain a mutant with high editing efficiency and easy screening, to study the effect of the dmrt1 gene on sex determination and differentiation of pelteobagrus fulvidraco, and to solve the problem of breeding a new pelteobagrus fulvidraco germplasm by genome editing.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a CRISPR/Cas9 system for knocking out a dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco, wherein the CRISPR/Cas9 system comprises the following steps:

(1) the target site 1 is designed on the first exon of the pelteobagrus fulvidraco dmrt1 gene, and the target site 2 is designed on the third exon;

(2) designing a primer to detect the accuracy of the target site in the parent fish according to the target site sequence in step (1), amplifying the target site 1 and the nearby sequence by using dmrt 1E 1F and dmrt 1E 1R, and amplifying the target site 2 and the nearby sequence by using dmrt 1E 3F and dmrt 1E 3R;

(3) using pUC19-gRNA-scaffold plasmid as template, performing PCR amplification of gRNA1 fragment with dmrt 1E 1gRNA F and gRNA R, and performing PCR amplification of gRNA2 fragment with dmrt 1E 3 gRNA F and gRNA R; taking the PCR product as a template, and carrying out in-vitro transcription and purification to obtain gRNA;

(4) in vitro transcription synthesis of Cas9mRNA by using pXT7-hCas9 linearized plasmid as a template;

(5) micro-injecting Cas9mRNA and two gRNAs into a pseudobagrus fulvidraco one-cell-stage embryo;

(6) and detecting the mutation type and calculating the gene editing rate.

The sequence of the target site 1 in the step (1) is a sequence shown as SEQ ID NO. 1; the sequence of the target site 2 is a sequence shown in SEQ ID NO. 2.

The sequence of the primer dmrt 1E 1F in the step (2) is shown as SEQ ID NO. 3; the sequence of the primer dmrt 1E 1R is a sequence shown in SEQ ID NO. 4; the sequence of the primer dmrt 1E 3F is the sequence shown in SEQ ID NO. 5; the sequence of the primer dmrt 1E 3R is shown as SEQ ID NO. 6.

The sequence of the primer dmrt 1E 1gRNA F in the step (3) is a sequence shown as SEQ ID NO. 7; the sequence of the primer dmrt 1E 3 gRNA F is a sequence shown in SEQ ID No. 8; the sequence of the primer gRNA R is a sequence shown in SEQ ID NO. 9.

The gRNA purification method in the step (3) is a LiCl precipitation method, and the method specifically comprises the following steps: adding 1ul of 0.5M EDTA into a gRNA in-vitro transcription system to terminate the reaction, adding 2.5ul of 4M LiCl and 75ul of precooled absolute ethyl alcohol to carry out precipitation, centrifuging and collecting a precipitate; adding 1mL of precooled 75% ethanol to clean the precipitate, centrifuging, collecting the precipitate, and removing the ethanol; 50ul of nucleic-free water was added to dissolve the gRNA precipitate, and the concentration was measured by Nanodrop and stored at-80 ℃.

The precipitation is specifically precipitation at-20 ℃ for 16h or precipitation at-80 ℃ for 2 h.

The centrifugation temperature is 4 ℃, and the centrifugation time is 15 min.

The step (5) is specifically as follows: cas9mRNA with the concentration of 600ng/ul and two gRNAs with the concentrations of 120ng/ul are mixed uniformly according to the volume of 2:1:1, and are injected into the embryo of the Pelteobagrus fulvidraco in the one-cell stage in a micro-injection mode, wherein the injection dosage is 1 nL.

The step (6) is specifically as follows: collecting 20 tail of the film-removed fry, and extracting genome DNA; amplifying a target site and a nearby sequence by PCR, directly sending a part of PCR products to Sanger for sequencing, and preliminarily detecting the gene editing effect according to a sequencing peak diagram; recovering and purifying the residual PCR product, connecting the PCR product with a PMD-18T vector, selecting positive single clone, sending the positive single clone to Sanger for sequencing, determining a specific mutation type according to a sequencing result, and predicting the change of an amino acid sequence; PCR amplifying the mutant and wild type genome with the pair of primers dmrt 1E 1F and dmrt 1E 3R, agarose gel electrophoresis detecting whether long fragment deletion mutation occurs; and calculating the gene editing efficiency.

The gene editing efficiency calculation formula is as follows: gene editing efficiency ═ 1- (1-a) × (1-b), where a is the mutation rate at target site 1 and b is the mutation rate at target site 2.

The second purpose of the invention is to provide the application of the CRISPR/Cas9 system for knocking out the dmrt1 gene at the double gRNA sites in the pelteobagrus fulvidraco in researching the effect of the dmrt1 gene on sex determination and differentiation of the pelteobagrus fulvidraco and molecular module breeding of high-quality parents of the pelteobagrus fulvidraco.

Compared with the prior art, the invention has the following advantages:

1. according to the invention, two gRNA target sites are designed aiming at the first exon and the third exon of the pelteobagrus fulvidraco, so that the knockout efficiency is very high.

2. The invention generates the mutant with deletion of long fragment of about 3kb, and the mutant can determine whether the knockout is successful only by PCR and agarose gel electrophoresis, thereby greatly reducing the subsequent identification cost.

3. The invention provides a mutant model for researching the function of the yellow catfish dmrt1 gene, and provides a new method for creating new germplasm of the yellow catfish; a yellow catfish microinjection system is established, and an effective technical means is provided for genome editing and breeding of the yellow catfish.

Drawings

FIG. 1 is a structural diagram of the target site of the molteobagrus fulvidraco dmrt1 gene;

FIG. 2 is a graph showing sequencing peaks near the target site of the dmrt1 gene in wild-type Pelteobagrus fulvidraco and the mutant;

FIG. 3 is a graph of mutation types to account for possible amino acid sequence changes;

FIG. 4 shows the result of PCR detection of deletion mutation of long fragment.

Detailed Description

The following claims are further defined and explained in connection with the detailed description of the invention, but are not to be construed as limiting the claims in any way.

Experimental reagent: the primers used in this example were synthesized by Guangzhou Tianyihui, and the Sanger sequencing involved was sent to Guangzhou Tianyihui for sequencing; the pXT7-hCas9 plasmid and the pUC19-gRNA scaffold plasmid were derived from the literature: chang N, Sun C, Gao L, et al, genome editing with RNA-guided Cas9 nucleic in zebrafish elements cell research,2013,23(4):465 Tissue DNA Extraction Kit Tissue DNA Kit and Gel recovery Kit Gel Extraction Kit from Omega; premix Taq for PCR amplification was purchased from Wuhan Pongziaceae; PCR product recovery Kit PCR clean Kit was purchased from Axygen; XbaI endonuclease and PMD-18T vector were purchased from Takara; the gRNA in vitro Transcription Kit, Transcription Aid T7 High Yield Transcription Kit, was purchased from Thermo; cas9mRNA in vitro transcription Kit mMESSAGE mMACHINE T7Kit was purchased from Invitrogen.

The full-length sequence of the yellow catfish dmrt1 gene is shown in SEQ ID NO. 10.

Example 1

Designing a target site:

the genome DNA sequence and the mRNA sequence of the pelteobagrus fulvidraco dmrt1 gene are inquired on NCBI, and the full length of the pelteobagrus fulvidraco dmrt1 gene is 18952bp, and comprises 5 exons and 4 introns. A target site is designed for each of the first exon and the third exon, the target site sequence is shown in Table 1, and the structure diagram of the target site is shown in FIG. 1. And (3) carrying out Blast comparison on the target point sequence on an NCBI website to verify the specificity of the target point.

The principle of target site selection:

A. the target site comprises 20 bases, wherein the 5' end should be GG, because the T7 promoter is adopted for the in vitro transcription of gRNA used in the invention, the first two bits of the transcription initiation site of the T7 promoter are required to be GG, and the third bit is preferably G or A.

B. The PAM region is composed of 3 bases immediately 3' to the target site, and the sequence is required to be NGG (N is an arbitrary base).

C. The target site is preferably chosen in the first 2/3 region of the gene CDS and after the ATG, but not in the last exon, and is preferably disrupted for important domains.

D. The target site may also be selected at the junction of an exon and an intron to disrupt splicing of the gene.

TABLE 1 target site sequence of Pelteobagrus fulvidraco dmrt1 gene

Step (2) target site confirmation:

a. for parent fish for artificial breeding, tail fins were cut, and genomic DNA was extracted using Tissue DNA Kit from Omega.

b. Designing a detection primer according to the principle: the forward primer and the reverse primer are both more than 100bp away from the target site; the PCR product is a single band and preferably does not exceed 500 bp. The PCR reaction system is shown in Table 2, the PCR reaction conditions are shown in Table 3, and the primer sequences are shown in Table 4, wherein dmrt 1E 1F and dmrt 1E 1R are used for amplifying the target site 1 (on the first exon) and the nearby sequences, and dmrt 1E 3F and dmrt 1E 3R are used for amplifying the target site 2 (on the third exon) and the nearby sequences.

Table 2.PCR reaction system:

2x PCR mix	25ul
		forward primer	1ul
Reverse primer	1ul
		Genomic DNA	1ul
Sterilized water	22ul
		Total volume	50ul

Table 3 PCR reaction conditions:

TABLE 4 primer information used in the experiments

Synthesizing gRNA:

a. gRNA primers were designed and synthesized, and PCR amplification was performed using pUC19-gRNA-scaffold plasmid as a template, with the PCR reaction system and the PCR reaction conditions shown in Table 5 and Table 6, respectively. Primer sequences are shown in table 4, wherein, dmrt 1E 1gRNA F and gRNA R are used for amplification of gRNA1, and dmrt 1E 3 gRNA F and gRNA R are used for amplification of gRNA 2. The base sequence of the front 17 th site of the forward primer is a T7 promoter, the base sequence of the front 18 th site to the front 37 th site is a gRNA target sequence, the base sequence of the front 38 th site to the front 57 th site is a pUC19-gRNA-scaffold plasmid upstream framework sequence, and the reverse primer sequence is a pUC19-gRNA-scaffold plasmid downstream framework sequence.

b. After the PCR product was detected by electrophoresis, Gel Extraction Kit (Omega Co.) was used to recover the Gel.

c. The gRNA was transcribed in vitro using the Transcript Aid T7 High Yield Transcription Kit from Thermo under the condition of RNA-free.

d. gRNA was purified by LiCl precipitation. The method comprises the following steps:

(1) the reaction was stopped by adding 1ul of 0.5M EDTA to the in vitro transcription system of the previous step.

(2) 2.5ul of 4M LiCl and 75ul of precooled absolute ethanol were added and precipitated at-20 ℃ overnight or at-80 ℃ for 2 hours.

(3) Centrifuge at 12000g for 15min at 4 ℃.

(4) The supernatant was discarded and 1mL of pre-cooled 75% ethanol was added to wash the pellet. Centrifugation was carried out at 4 ℃ and 10000g for 5 minutes.

(5) The supernatant was discarded, and the centrifuge tube was placed in a fume hood to volatilize ethanol.

(6) 50ul of Nuclear-free water was added to dissolve the gRNA precipitate.

(7) The concentration was measured by Nanodrop and stored at-80 ℃.

TABLE 5 PCR reaction System

TABLE 6 PCR reaction conditions

Step (4) synthesis of Cas9 mRNA:

a. digesting the plasmid pXT7-hCas9 by XbaI endonuclease, incubating for 5 hours at 37 ℃, taking 1ul of digested plasmid and undigested plasmid respectively, detecting whether the digestion reaction is complete by agarose gel electrophoresis, and if the migration rates of the digested plasmid and the undigested plasmid are completely different, indicating that the plasmids are completely linearized. The cleavage reaction system is shown in Table 7.

b. The digestion product was purified using the PCR clean Kit from Axygen to obtain linearized plasmid pXT7-hCas 9.

c. Cas9mRNA was transcribed and recovered in vitro using the mMESSAGE mMACHINE T7Kit from Invitrogen under RNA-free conditions.

TABLE 7 digestion system

10x reaction Buffer	5ul
		BSA	5ul
XbaI	2ul
		pXT7-hCas9 plasmid	30ul
Sterilized water	8ul
		Total volume	50ul

Step (5), microinjection of pelteobagrus fulvidraco embryos:

cas9mRNA and two gRNAs (dmrt1gRNA1 and dmrt1gRNA2) were mixed at final concentrations of 300 ng/. mu.l, 30 ng/. mu.l, and 30 ng/. mu.l. About 1nL of the mixture was microinjected into embryos of the pelteobagrus fulvidraco in one-cell stage.

The artificial breeding method of the yellow catfish comprises the following steps:

firstly, artificially hastening parturition. The artificial oxytocic of the yellow catfish is generally divided into two needles to inject oxytocic hormone, the needle pitch is 10 to 12 hours, and the hormone is dissolved by medical normal saline and then injected. First injection ripening: injecting luteinizing hormone releasing hormone analog (LHRH-A2)15-20ug per kilogram female fish; and (3) hastening parturition by a second needle: LHRH-A215 ug, Deodourone (DOM)10mg and Human Chorionic Gonadotropin (HCG)800 International units were injected per kg of female fish. The male fish were injected with a second needle at doses 1/3 to 1/2 of the female fish. The temperature of parent fish induced spawning water is preferably controlled between 28 ℃ and 29 ℃, and the induced spawning effect time is generally 15 to 30 hours.

And secondly, artificial insemination. Preparing a clean anhydrous glass culture dish, grasping the female fish with the left hand, wiping water and mucus on the fish body, slightly pressing the belly of the female fish with the thumb of the right hand, and extruding the roe into the culture dish from the genital hole; killing male fish, taking out spermary, cutting with clean scissors, adding 2mL of physiological saline for dilution, collecting into a clean EP tube, and keeping in dark at 4 ℃; 50ul of semen was added to the petri dish containing the roe, and stirred with clean feathers for 30 seconds to allow the semen to be in full contact with the roe, and then spread in a glass dish containing clean saturated water. After five minutes, the fertilized eggs were stuck on a glass dish and washed several times with saturated air water. Twenty minutes later, the embryo develops to the first cell stage, microinjection is started, and the embryo develops to the second cell stage, and the microinjection is stopped, wherein the microinjection can be carried out in about 30 minutes.

And (6) detecting mutation types, and calculating the gene editing rate:

a. and (3) hatching the fertilized eggs in a water body at 28 ℃, taking out the fertilized eggs from the membrane after about 72 hours, collecting 20 tails of the small fish fries with the membrane, extracting genome DNA, and adopting Tissue DNA Kit of Omega company.

b. The genomic DNA was subjected to PCR amplification using two primer pairs, dmrt 1E 1F, dmrt 1E 1R, dmrt 1E 3F and dmrt 1E 3R, respectively. The PCR reaction system and the PCR reaction conditions are shown in Table 8 and Table 9, respectively.

TABLE 8 PCR reaction System

2x PCR mix	25ul
		Forward primer	1ul
Reverse primer	1ul
		Genomic DNA	1ul
Sterilized water	22ul
		Total volume	50ul

TABLE 9 PCR reaction conditions

c. After the PCR product is detected by agarose gel electrophoresis, one part of the PCR product is directly sent to Sanger for sequencing, and the gene editing effect is preliminarily detected. As shown in FIG. 2, the sequencing peak pattern shows a nested peak after the target site, which indicates that insertion or deletion mutation occurs, and the gene editing is proved to be effective preliminarily.

d. The remaining PCR product was recovered and purified using the PCR clean Kit from Axygen. The purified product was ligated with PMD-18T vector overnight at 4 ℃ (ligation is shown in table 10), transformed into competent cells, plated, single clones were picked, positive clones were PCR detected, 20 positive clones were selected for Sanger sequencing, mutation patterns were statistically calculated and amino acid sequence changes that may result are shown in fig. 3.

The specific conversion steps are as follows: the ligation product was mixed with 50ul of competent cells and placed on ice for 20 min to mix well; after being heated in water bath at 42 ℃ for 90 seconds, the mixture is quickly placed on ice; adding 800ul of precooled non-resistant LB culture medium, and incubating for 50 minutes at 37 ℃ by a shaking table; centrifuging at 4000g for 2 minutes, discarding redundant supernatant, reserving about 100ul of supernatant, and resuspending the precipitate; ampicillin-resistant plates were evenly coated with the bacterial suspension and cultured at 37 ℃ for 16 hours.

TABLE 10 connection system

Solution I	5ul
		PMD-18T carrier	0.5ul
Purified DNA	4.5ul
		Total volume	10ul

e. The genome in step (6) a was PCR amplified using the pair of primers dmrt 1E 1F and dmrt 1E 3R, and agarose gel electrophoresis was performed, as shown in FIG. 4, the mutant detected a band of about 400bp, while the wild type did not detect it, indicating that a long fragment deletion mutation may occur. The PCR product was recovered and purified, and subjected to Sanger sequencing, and the sequencing result was aligned with the genomic sequence of dmrt1, whereby it was found that the genomic sequence of about 3kb including the intron was deleted between the two target sites.

f. Calculating the gene editing efficiency, wherein the calculation formula is as follows: the gene editing efficiency is 1- (1-a) × (1-b), wherein a is the mutation rate of the target site 1 of 62.5%, and b is the mutation rate of the target site 2 of 83.3%, so the editing efficiency of the gene of the Pelteobagrus fulvidraco dmrt1 knocked out by adopting the invention is 93.75%.

Sequence listing

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ggacaataga gagcaaagtg gcacttgctg gtcatcttct gaaaccaggg catagacggc 2940

acattccaaa cctgcacatg agataaaatg ggaggtgaaa agataacaaa aagacagcaa 3000

tgacgcattg acgcattaac aaggaataat gcaggaatag tttaatagca gggtcactgg 3060

tatgtttcac cagcagatgg ggccataagc tatgacaaca ctgatcttca tcgcttgtaa 3120

aactgagaag acaaagatgg catggaattt atcataaaaa tttgaaatac atatattttt 3180

caaaatcttt tgaattgact ctgattccct gagcttttag gttgtgtaat tattcatgac 3240

caataaattc agtttacagt ggtttgaatg gctctgtaag tgtaagatgt gttgtttcag 3300

ggagtcgctc ttcattgtcg cagagcccca ctgcagccag caggggacac tctgagggct 3360

ctgctgacat ggtggtggat gcctcctact acaactttta tcagcctacc cgatacccag 3420

cgtactacag caatctctac aattatcaac agtaccaggt aacaacacac atatgcagtc 3480

acattataca ttagaaatgt catacatatt cggtacactc tcagaaatat aataataaac 3540

tatacctttg cttgtaggat agtgtatctt tcttactcat aatatactca cagtatcttt 3600

catctggaaa tttttttata gtgtaacttt ataagggtat ttttgttaca ttaaaagact 3660

gtaattaaaa agaaacataa aagaaaaata caaattagta actagattgt atattacatg 3720

cagcatttaa tctgtttgaa ttaagccctt ctgatacccc ctgcaaaata atgtgttaaa 3780

acagcagcaa catgatgttc ctacattgta agttttatat agtaatcatt gctgcagaag 3840

ccataaccat tctacatagt attaatagat tagaagtatt atagtcatgt aaaatgaatt 3900

aagatttggg gagttattgg tgggaatatg tttaccatta aacacagtgg ctgcacattt 3960

tatgcagagt gagtgaatag aattggttag ttaatagaac agtagtgttg acatgacttt 4020

gtgtctggtt tttccaatcc cacttaaaaa tccactccaa acaggaagga ttgctccata 4080

ttaactcaac agccatgaag tagaccacca tggtacactc attttcccac tcacctaagc 4140

tgtgtcttat gtggtccagc agaatgtggt aatgagctga ttaagtgggt tagagcgctt 4200

gtttatacgg gttcctgtca gtgaacccag ctgcctgttc taacttggct cggtggcaca 4260

gtgagcattt acgcatagtg cctgtcctcc cgttccgagc cagtggctga gtacagcaag 4320

gaaggtgtgt agtcttcatg ccacgctgtc tcttttacaa attgtttact ttaccatgct 4380

tcccccagtc ggaactgcaa ctaatgctgc cactgggagt tctctaaccc ttatccaagc 4440

catgcaattc cactgaattt tgccacctta agctactcag tttggcaaaa gattttaatg 4500

catatcttta aatgcttgag gtggagttga tattgctttt gttctctggt ctgttagact 4560

ggagctgcaa aagcttcaaa caggagaaaa atatcttact tgctttaaag atcagatatg 4620

tcctatacac gtaattttac tgacagggtt aggcagggtt acactcctta agtttgcaca 4680

ttttacacat gtaatgttaa tattttgtgt gtttctcaac accataaaaa ttccttgtat 4740

gtatgagcat atttggcaca tttggcaaat gaggcacatt ctgattaagt gttagattta 4800

taactatata taacatatac aaaatgccag caacttataa taagggattg tagtgaaact 4860

ggttcttatt tttaataggc tctgtgatat agagcaaaga cgagtgtttt cacaaggatg 4920

agtctggtgg agtaataatg gtattattaa taatgataat atatgtttta tatgaatagg 4980

ctgtaaaggt atgacatatg atcctccgat gtaggtagtg agatggacgt agatttgaat 5040

aagatcgtga tccatgtata taatgtttga agacagtttt tttccatcac tgtttaactg 5100

ctgtacaaca aataaagagc aggttcttgg ttctaagaat tctgtttatt tttattcact 5160

gacagttaca gtgatctaaa agctaaaggt gtctaacttc taattaaggc acaatttcca 5220

ttgcgttaca taatgttaaa ggtttccatt tactcatatt gaatcggaca atttgaaatt 5280

gagggcatcc tcaaagtcat ggcccttcag ctttgatatc ccatcaactt gagacacttt 5340

tattaacctt aatcaatcag ccaagatatc tctgacatat gaggcactgt tcacacggtg 5400

acatgattgt agtatggaga tcatcctgta tatttactct gatttatgtt ataaagtatc 5460

accattgtat cctggatgat ttatccatgt gtgaaataaa ttaaaccagt atttcttctg 5520

ggacttaaaa atgactggta tgtccatgtg ggctgttgat ggagccatat ttcattatgt 5580

atgtgcaata gggtgcctga cagcaacatc ctgcaccatg gtcactgctt tcgtcacact 5640

tctttgtgcc atttgaattg caaaggttaa tgatattgct atagtgtttc agcacgcttg 5700

aatactttaa tgttgatttc attatgcata atggcactac ctgctagatt gcaaggattt 5760

attttctcag catgttgcat tgtaaacaaa gatggaccag atctgagctg atgttgcttt 5820

ggtagaaccg atgtttctta ggtgttaaag ctgaagatac agtagtagcc ttaattttgc 5880

aagaccttat gttatgtggt gtttttagca ttcagcttta ccagtcagct gtagacattt 5940

aatttttgct attgtgggca tgacaaaagc tttgggtatg ttgcattcag cctgcgcatt 6000

catatttaag ttttatgaaa tcctcctact aaatcttatt aaatccattt ttaataatat 6060

gcatttcctt aagaatctaa tccgttacca tggtctacaa tccttaggtg ataggttgat 6120

agaaatcggt ttagcgttgc tgtaggggaa aagttagtga atgaagaaaa ttctccccag 6180

cttgtcttta tctaagcacg tccatctcct gccctctacc atctcaagca aacagggctg 6240

cgctgcactg caggtggaga atgaaaatat gagggtggcg tagaggatcc agatagctga 6300

tggtgccaga taagagccct ctgagggagg taagggagat gagccagccc ctattaatgg 6360

gtgctgcact gaagctttca cactaaatgt agcactgcac ccacacgtta atacacaatt 6420

gtaactttct tcctgttgaa agggaaagtc ggactttaac atatatacac aaataaattc 6480

ctacatgaat atgatgtaga gagatatgaa ttttcattat atatttgaaa gatttgtctt 6540

gtgcttaaac tgaacatatt acctttgtac atgagtgtga gaggtgtcaa atacaatact 6600

aatactaatg aatgagggca gtttaagacc atgaggccga attagtctgg caaaaaattg 6660

aattggatta acatgagcat tgaaatcgaa ctgtgcctta aacagtcatc ttcaattctt 6720

gttgtgacag atttcaaatg cttagccatg tgtatttctg ttttgcagtg catgtttcag 6780

tggagcatgt ccagaaatat acagttcatt tttttgttgt atttgtcata ggcaatagga 6840

gaagcccagt gtagattaaa taacagttta taaagctgtc tgggattgat ggggtgaaca 6900

ataaatggtg acatattact ggctgacaga aatcaaattc cataaagcta gaattgaaaa 6960

accctttaag ctgctttgtt gctgactaga cagctcattt taaagttctc acaagacggt 7020

agaaaattga cctgagatgt gttgctgttt attttgaggg aataacttat caggtctgag 7080

aagccaggtg agactcgtaa gggctgtaga ctatatcttg cgttttttat ttcatgatga 7140

gacatgtaca actttgtttt aaatagcagg cttgttttca gaatttctaa tttgtgttgc 7200

attgttttgt atgtactatg atcaagatgg cttttttttt ttaaaaataa aggtgatttg 7260

agttgatcgc ccttgtataa acatatacat atgagctcca gaattattgg caatcttgct 7320

taaataatgt ttgtgtgtta attagctgga tgtatcagag tattataatc aatatttgta 7380

aagtaaatct gacgtttgaa gtaaatctga attctaagaa aattttaaca gaacatagat 7440

aaacatgaat catatattta attaaactct atgcatcaca ataattggca cctaatcatg 7500

ttggcatttt atgtttcctt ctgccatcac aacagtactg agtcttattg tacaattgaa 7560

ttgagtcgaa gacatttgag aatacagagc aaggttctat aagatcacat cgtagtatag 7620

aattcagatc ctctagggtc ttactgtagg tcctttcttg tgatatgtcc tcttaaacac 7680

agtagacttt gatggccaac agctaagatc actgaaacat ttatacatgg aattcattgt 7740

atgcttagga tcactgtcat gttgaaagat ctacacatga cccagttgta atatcttggc 7800

agaggcacat tttcatttaa aatatctggg taattggtaa ctggtacttg accaagtata 7860

taagggtctg ttgagagaaa gaaaacagtg ccatagaacc tctgctataa ttgaaagcca 7920

ggattaggtt attttttata tatatacaca ttctttttta tgcagaaccc accactagat 7980

gttgttgcca aaaaagtctt atttgaccat agtacccagt ttcagtgagc atatttcatt 8040

tgcattttta ttgagaaata ctagggctgc tgtacattga catggattta ttctaaaata 8100

aattttaggt ggaataatta tggagataac tgtattcata tttcatattt ccagttctat 8160

taatcttaca acgtggtggg tgtttgtatg tctgtgtctg tacgtgaatc atttcacttt 8220

gaagcagatg cccagtggag atagccgcct gtccagccac aacatgtccc agcaataccg 8280

tatgcactcc tactattctg cagcttccta tcttagtcag ggcctgggca ctgctacctg 8340

catgccaccc attttcacca tggaggacaa taacatctgc cctgaggcca aaactgcagg 8400

tcagtgtctt catcattctt ctaccccttc ctcctttctc tctcacatac acacatgcca 8460

ataccactca caggcccata aagttttatc cagagtttat caacagcgtg cacaaagtac 8520

atctgaggga ggctcactct gcaagtcaag agcgccataa aattcacaca aataaataaa 8580

ttcaaaagta aaacttttat tatagctatt tgttcttagg ccttggaatt taaagtgaca 8640

tgagctgcat ttacaagtgc agaagtcaca cttttttaat actttagttt cttgttgcac 8700

ctatcataaa tgcacacaaa tgtctggttg atgaactgcc cagagcagct caatttatga 8760

acaaatgctt gagaagtgat gttgatcaac atcttgcaat cttttgtatt ttattagacc 8820

caatgttccc tcatctcttg aaagccttta cctgtttgtg ttcttgatga attttacatg 8880

ctgcttaccc atgagggtgc ctgtccccct gcactctcac tccagggtgt cctacgctcc 8940

attacagcac aaaagcactc cagtctgggt ggtctcaaaa cattgatatt aactacccag 9000

ttgctggagt gatggctgtt tcctgtgact agcattaaac aaataatatg aggcttgttt 9060

gcaccagtag taaaaaatcc agtgctcaac aaagtgatct ataaagatta aatccctttg 9120

aattacaaat tcaggctatt atagtttagt gtttagatga aaattattta ttgcaaattt 9180

tttatttact tatgaaaatc atcatacatt ttacacatcc agtatgcaca taggaaatgg 9240

catggtatct ggtcattgtt tgccacagaa gccagctaga taaccttcct aatgttagta 9300

atgcataatc tggctgtcat ggtataattt gacaggtgta gctattgtta aaaacccaca 9360

gagaatatga cagtatactt caaggcttat acggaaacct gtgacaaatt actaatttac 9420

aaaaattaac ttcacagcta gcatagaata tctggttgac actaccacat ttctagtttg 9480

cgtaatcatg tagtatttag aaaaaaatac aggtaaagag atgtcctatg ccctattaat 9540

tgttttgtct tgggctttaa aacacttaac atcgttggat tgactagatt ttagaattgt 9600

ccagaggtac tttgaataac cttttaggag attatactga ctcagacgat gagtattaca 9660

ggagtattat taaaataata agccgttttg tgcttgtctt ctcccatgct actctgttta 9720

gatggtctta tttggaatga catggccatg accttgcatg catggccagg ctggcatggc 9780

cagaaaaagg ctgttatctg caaacggttg ctgcctgttc tagtgtggtg cacatagatt 9840

tcacattctt tctatcatca ttattagtac agggtacagg cagtacaact aggcttgatt 9900

agctatttag attagattcc tgagcacagt tcacaagtta ttgcacaagc cgacaagaga 9960

ttatgttctg ttcactatgt ctatctatct atcgatctaa caatctgtca gtctgtctat 10020

tctatatttt ttagatggaa gacatttgag ttggagatca aaagatggat ttgagacaat 10080

agatcagatt ataagatttt attccctgat atatatgtaa aacaacaagg aaatttcacc 10140

ttttgtttga atccacccat ctgttgtcag atgatcaata atattggaac atatgacagg 10200

tatatcttgt tgcccagatg tgccctgtta aattgatcgt ttaaacaatt aacatttcga 10260

aggttagaga agagagatca ttgcacaagc attgtttaaa agatttaaat ggtgtaaata 10320

agtaacagtt cctctccaga tttaattttg atactgcaga aaacgtctga tctttggtcc 10380

ttaagatgta atgctcttgt aaacatatgt ctttctatga gaaggcttgt gagtgcccca 10440

tacacctgct gtgcctcctg tgctcccacc ctgcacaatg tgtatttatt tcccccttgc 10500

tctgtggagt ggtgagggtg gagtgggggg aagtaatcag ttggccaaat atgaggccaa 10560

ataaccttca agaatgccat gctgtccttt ctgagcagcc ctctctgccg gtgtctcggg 10620

aaaattgcct ctggggttag tgagcgggaa cagttaagat ttacaaacaa tgatgaagct 10680

tgtcatgcgt ctttctccat ccgtcacgag tggcggccct gccggggcgt ccaagtgttc 10740

ttcataaaag ccagctcgct gtgaaatacg cccctgtgcg cactttactg ccgcgggctc 10800

gtcagcagcg ctttgctgta aatctccacg ccactagtga caccacactc catcacccgc 10860

ctggggttgg cttacatcca ccccacatct ctctcccaac ccatcctcct gccccgtcat 10920

gctctgagat gatgtgctcc ctgtgagtta tattggaatt ttagtgttgt atgagacagg 10980

ttttaaccat ttactttctt tcttgttttt gttgttgttg aatattgcta aaagtttcat 11040

gtaggaaggg aataatgtct tatgcagttc taaaaagaag gacaccagtt agtataccat 11100

gtagtaagca aatttagaaa gtaaatcttt aagttttagc agtcatttat ttgggtttta 11160

gtacttttgt caagtttaca aattcaatac attttaatct caagttttat tttttagtat 11220

tagaacgtgt aaagcttttc aaggctatgg atatttattg atcttactgt tctttgtgct 11280

gtgtctctaa gccacaaggg taatggatgc atctcattat tattcacatt tgaacttgtt 11340

aaagtaagac tcctttctgt gcaaggaatt tgatatttgt attcatatca attgcttgaa 11400

gaaatgctga ataaaagcca ttttgtttag tcatttcaaa ttctaaaata agcttataat 11460

gacaaattta caaaaatttg aaaattgtta ttctctcaga tcatccctca cagttgatgt 11520

ggctaaagaa ccagctgttc ttcacagatc atatagaact aaaatgtaag tgagttgacc 11580

tcagtgttaa aagatgtatt cacaatattt tccatcgcta agtagttgcc ttcgctgttt 11640

tcccattttc ccgtttgaca ttacaagttt aattcaagat cggtattttt gattactcat 11700

cttatcaaaa aaacctttat tttaaaaatg tatagtggaa caacacatta tgaaactgct 11760

taatataatg aattacttaa agtttcagtg tagtctcaac tcagattaca tgggaaccaa 11820

caaaaacaca ggttgtgaca aaataagtac ttgtagatca agtacaaaca aactacattt 11880

ctggaaaaag acagtttcac aataatcaaa ggccctattc actctttcag attatttcaa 11940

caatgaaaac tttctctaaa tgctgtgaag tgtgaaataa ttcaacagga aaaggtgtaa 12000

cgtctcatgt aacatagaag ccataccgct tttgtcaaca tgactaatct attctcttga 12060

catcacacct agcagagcag ttaactataa ttcgaactca gaaggagaaa tttttttctg 12120

taaattgaca gtctgttatg tatatttggc aagatttagg atgtatggaa atttgttttg 12180

cgtttgcata aatcagtcct gcaagcaata ttaattacgg aacataatat tttattgttt 12240

aattagctgt gttttttagt ctgattattc ctgctaagat aatgggcatc ttactggcag 12300

ctctgtggca tttttcatgt ttacattagc acagaggtta gtgcactgtt ggatatattt 12360

gttaaattat ttacatccct tattttccta agttagttag ataattttgg tgtctagatg 12420

agtggatatt atcaatggaa gtgcttgtat tgctgagtta tgatatctta tatgctgtca 12480

tccatattct gttatttggt tttcaagctg gaaaattatt gaagcacatg attctatttg 12540

ccaacatatt acacttctga atcaaacatg atatacacaa tgtggtcagt agtttgttgt 12600

cacctgacca tcacacccat atatgatttt tgaacaccac atttcagatt tattcccttt 12660

tcttttggtt atattaaagt acactcatct ggtaaggctt aacaatagat gtggctgtgg 12720

gattttgtta ttcagttagg acagcttaag tgaggtcagg cactgacgtc aggtgaggag 12780

acctggggtg aagtttgtgt tctagttcat ccaatttaga agctgcaaaa tatggtatgc 12840

atgtttttcc ttttgattaa taatgatttc tgttgacttg tatgaggagt taatttacta 12900

tagttaaatg tagcatttta tggatacaaa ataaacacat caaaccccaa atgacactct 12960

tttgttagag tgggcctgga ggtgaaatcc atgactggag tctaactaca gtttgctgtc 13020

taaaagcttt aggcacacag tgactcctcc atttcatgct gtcttgccct ctctcttgct 13080

gatgctaaat gctttgtctt ttctctctct atggattggt taaataccag tgtaattgtc 13140

tgctctggtc tgaagcactg ttgtaatgtg ttgtagaaca tcaccattaa gacttgcctg 13200

aggcattgga gagattaaat gtgtaaggag cattgcttta tgttcctcac cagccattgc 13260

cactgtccag tgctcagaat ggttgagcca ctgggacaca gacatattga aaagagtaga 13320

tactaaagta gagactggcc aatataacta gatatcaatg gaaatggaga aaatttgaga 13380

ttactccctt gataaaccaa gctagaatgg ctgtttcatg caaatgttta aaaaacgcat 13440

ccaagtaggt ttaatatatt cttaatgttt gtagttaagc ctatgatctc atagcacaca 13500

aagtgagtaa gggataagag ctgaggaagt tataagcttc gaaaaaggta gctttgtgtt 13560

taattttaaa ccattgcaat gttagtgtgg ctatgaaaag cttcttttgt atggttctga 13620

tcaattctct gtctgacttt ctgacattaa gagcccaatc tgaatgtaat gacacactta 13680

tttcaccctt gggagtgtag tctgtgctcc accccatgac gctatttttt tcacatctct 13740

aattccacat tcacaatttt aacagaatga caagaaatcc ctcaaagcat tgattgcatg 13800

tctcaggtca ttatagtata tctataatgt ttcgacagat cagagcatgt gtccagagat 13860

ggacaggtca ttttgtggaa gctggctgag tgccagtgat ttagcatggc ggtgggcctg 13920

tcaattctct caatgacact ctccggctgg ggacagattg gttaacaatg cgggactgga 13980

ggtgcttatg tgtttaatta gcctctgacc ctcacttgat gccttctgtc agacactact 14040

ggccactcat gctattctct aatagtccaa taatgatcat tgaatgtgtt ggttccaata 14100

ggtagtcatg aaaatatagt gatgcatttc aactgcttgg tcctctttaa gaattgtatt 14160

ttgacttcag ttatcagaga cattttatga tgatttgttt tagaatttac atagagtgca 14220

aatgtattaa tcacttattg gttttgtttt cagggtacta tttctgattt gtttggtcca 14280

taagcagtaa aagcttagtc tgtgagaatg tctctgttgc agctattgac tggtttgagg 14340

agtcagagtt tgtttaaggt ttaagtctcc actgcatttt ctgcttcata aagtcctgcc 14400

aaagctctaa tgatggagag tgatgcgcat gtctctcaat aagacagacc tccatctctt 14460

ttttgccctc atatcttcct tgccgcccta gaggacagct ttgcaaagtg cataactaag 14520

atttaattat tgtataaaac actaggttat gttagtgtag ctatattatg ttttaaaatt 14580

actaatacat gcatttatat aaattattat tattattatt tatttagtgt ttatttcatt 14640

gcttctttat ttacagctca gtattataca ggccttatat gtaagtacat ggcagagtat 14700

gtatatctgt gtttatctaa gtggagggat taggcaccct catggccctg tgcaggtagt 14760

ctatgagctc tggcagaaaa gccttatcca caatagatct ggcctaaatt accatcttct 14820

ctcacatctg ggcttttgtt cactttttgt tccgactgcc agaaatgatt gagctccttt 14880

caccttctcc cctccccttt tcctgggctt caaaaaaaaa aagacttcct tttgttaaaa 14940

cacacaaaga ctggttaata gtttggaaat ataagatatt cagttaaaca gaggtgaata 15000

gggcaaaagt tcacggaaga gaatacgttc tttctgaaca gaaagaaggg taaatcttct 15060

cttgtttcag tactgttcta cgcatgacag acgtgtacaa gtatttaaac aaacttgagt 15120

tcctgccaaa tctcagcttc aacatttttt gtaaccctgt ataccatacc aatgcctctg 15180

tatgaactgc atgatatcag atttagaaca ggaattattt gcttttctta aggttacagt 15240

atttcactct taactcagac atccatatat atatatatat atatatatat atatatatat 15300

atatatatat atatatatat atatatatat atatatatat atatatatgt gtgtatatat 15360

atattacatt ttttttgaac attgtttaac tgcttcacca tttaagaaca ttacaagtat 15420

tgtaaaacat ttgataggct tcacattcat ctggaattaa gtggcaatta ggtgatgatt 15480

atacagaaca aagctgtgac actggtcatg tctgcatgcc atgcaagccc tagtctgtta 15540

tttacctttt atacaaatga cctaccagtc accctcaagt gcttttgcct ttaattacac 15600

catagttttt cccccctgat gaactccttt agaattaaca cagccactaa ttcaaaatcc 15660

cttatctata tcacaccatg ttattgctgt tcacagagca tctctgcagt ttagctacac 15720

aaacattagt attaacgatt ggttggtttg gaatgggtga caaactgcag acagcatgcc 15780

aagtcctgaa gcctgaaacc aatcagaagg ttaatgagga gaaaaattgc tccttttttc 15840

accagtgctc cttaattacc tccatgccag agcagagcac tgggtagaag gaggagaata 15900

aaggcaagag tggcattgag atgaacttta tatcgtctct tgtgagttac gaacgtcagc 15960

tgaattagat ttatgtaatc cctcttgcat tgtaacttcc atagaccgca cagacactat 16020

agtatcagtt ttatatgtgc agttagttaa atactgaact gaactggctc agtgcacaag 16080

tcatgatcca tgtgtttata gtaatgtaga ttattctaat agagagtgct cagtgtttca 16140

aattaaaatt agcatcacag ataatttgtc aatactgtga gtgcaataaa accgaataca 16200

taaccaactg gaaaaaataa catttaacag ttatattctt attaattttc ttaccagtaa 16260

aatttccatt attttcctgc tagtgttttg ttgagagagt gttaaagaaa gctaaattca 16320

gttgatatat tgcttatcca gcactgcttg ttctacatct gtctgtactg tgcatgccca 16380

gcactcaata tgacatcaag ttcaatggtt ctgcccaagg ggcttgaact cacctcagcc 16440

tgtgtgattt atgtttgcga gcactttgtc tactttgttg taatgttgag tgctgtcact 16500

attataattg caaaaagctg acagattgca tgtacactga ctttcaaaac tgaggctgta 16560

aaaatgcttt cactcaccaa tttctcacat tagtatgtaa tctacaaggc atttgggccg 16620

ttttgcagat tttacagtaa ggaaaaataa acatgcaaac acttttgttt ttgttattaa 16680

acacttatta tattaaattt ctatctaaca attatcaaca cttaatgtgt gtgtgtgtgt 16740

gtgtttttgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt ataatacttt cagtacatat 16800

gttcatttca aaattccaca catgcacaca cagtgtagtt tttctaatat tagtaatttg 16860

tctcagagtt ttgttgactc attcttcttg gcaaatcatc ttattttctt caaaatataa 16920

gggtcctaca aatttagacc agcagtctct tacatcaaag ctcttatcac tccctgcact 16980

gcaccacact ccctcagatc ctctagcact gcttgactgg tcccaacctt tttcagggta 17040

caaggtaggt ctgcatcaag actcatctct gttctagcac ctaggtggtg gtatgaaatt 17100

cccataaatg tgagaacagc caagtcactg gctgacaaag gttagttaaa gagttacctt 17160

ttcctgaaat acataaacta gcacttgttt tatcattatt ttttaaattg tttgtgtgct 17220

tttcttgatg tattacctat caacagagtt ttagattgta gatcagtgat tcagtgtcaa 17280

tatattcact tataaagaca aagcactttt ttgacatcac tctgtataag ggcatctgcc 17340

aaatgccata catgtatatg gaaatactag tagttttgct gcattgttca catgtttgag 17400

cttctgtgct ttgcagcttt ctctgctgat ggcgttcctg acaccagctt ggcctgcata 17460

cccatcatca gccctatggt gagtgctgag aacaaggcag aatgtgagcc caactctgac 17520

tctggagcat ttactgtgga ctccatcata gagggagctg ccaagtaaaa gtctcagaga 17580

atataccagc tgcacttgac ttatctcatg gcactagggt attaaggcca tgtttttttc 17640

cctcacaaga tcagcattat atgctaaata agttgtgacc aacaagttta gattttcatt 17700

ataatttgtt tcagttcgat tttgcccaat agctgcagtt tttaatgact gaattttgtg 17760

aagtttgttg aaatatttta aaatttggtt ttagggcaaa ttacttaata ctttatattt 17820

cttcataatc atttagatgc cttctgcttt tttgaaatca caagacagtt aaagagtttt 17880

agtaaacaga agacctttta aatgtgtgtc tgatcacccc ctagaaaaag ttccttcaat 17940

gtttgcatgt gaatgcaatc tgcggcacat gtcagcttta taactgttat tattgcttta 18000

ataaatacaa atgtttccac cacatttttt ttaccattaa tgtaaagcaa atgagtttct 18060

cccaatgcta tgatattcag tctcagatca tttttagcac ctttcatcca gacaacaggg 18120

gatgcagtta tgggttagta tgcaggaagg aatactaatg aattatgctt caatctcact 18180

acaaacagtg cgttgtttat ttatggccac cagcaaaagg gcatcattta aatttggatg 18240

atttgcatca ctatctacct ccttgaattg tgagaggtgc tttaacacac tttagtttat 18300

aagctatata tgagtgcttc tgccctttct ttgcacaccc cagagttgta ctgtttgccc 18360

ttgaaaatat ccctgaataa tgtgtgataa cttgccttaa ttaaggaata tactctacac 18420

agcctcagat caaggcagta atttttatac attttgacat aataataata ataataataa 18480

taataataat aataataata ataataagta cacatatctg ttgcaaatag atctctgcca 18540

ttaacttaga cacagtttaa agaaaatata tattatccag caaaatattt tgtactatat 18600

aatttttata ctccagaaaa tgaaatatct tccagttaaa taaggcaagt tgaattacac 18660

atgcaagtca ttatttgtat attttcttac taataaagca agaagactga atcactcatt 18720

tattatgaag agtggaggtg ttaccatttt agacaatatg ttcatgttaa gatttacttc 18780

cttaattatt gcaacattga gttgtgagct gtaccacaaa ctggttctat ttctttagtc 18840

tctgcactgt gttactgtga ctgactgtta tggagattaa tttcaaatgc catgttacaa 18900

aagcggcagt tattgttatg tcttaataaa acagcaagaa agctcattta aa 18952

Claims (9)

1. A CRISPR/Cas9 system for knocking out the dmrt1 gene at double gRNA sites in pelteobagrus fulvidraco, wherein the CRISPR/Cas9 system comprises the following steps:

(1) the target site 1 is designed on a first exon of a pelteobagrus fulvidraco dmrt1 gene, the target site 2 is designed on a third exon, and the sequence of the target site 1 is a sequence shown in SEQ ID NO. 1; the sequence of the target site 2 is a sequence shown as SEQ ID NO. 2;

(2) designing a primer to detect the accuracy of the target site in the parent fish according to the target site sequence in step (1), amplifying the target site 1 and the nearby sequence by using dmrt 1E 1F and dmrt 1E 1R, and amplifying the target site 2 and the nearby sequence by using dmrt 1E 3F and dmrt 1E 3R;

(3) using pUC19-gRNA-scaffold plasmid as template, performing PCR amplification of gRNA1 fragment with dmrt 1E 1gRNA and gRNA R, and performing PCR amplification of gRNA2 fragment with dmrt 1E 3 gRNA and gRNA R; taking the PCR product as a template, and carrying out in-vitro transcription and purification to obtain gRNA;

(4) in vitro transcription synthesis of Cas9mRNA by using pXT7-hCas9 linearized plasmid as a template;

(5) micro-injecting Cas9mRNA and two gRNAs into a pseudobagrus fulvidraco one-cell-stage embryo;

(6) and detecting the mutation type and calculating the gene editing rate.

2. The system according to claim 1, wherein the primer dmrt 1E 1F in step (2) has the sequence shown in SEQ ID No. 3; the sequence of the primer dmrt 1E 1R is a sequence shown in SEQ ID NO. 4; the sequence of the primer dmrt 1E 3F is a sequence shown in SEQ ID NO. 5; the sequence of the primer dmrt 1E 3R is shown as SEQ ID NO. 6.

3. The system according to claim 1, wherein the primer dmrt 1E 1gRNA F in step (3) has the sequence shown in SEQ ID No. 7; the sequence of the primer dmrt 1E 3 gRNA F is a sequence shown in SEQ ID No. 8; the sequence of the primer gRNA is shown as SEQ ID NO. 9.

4. The system according to claim 1, wherein the gRNA purification method in step (3) is LiCl precipitation, and the specific steps are as follows: adding 1ul of 0.5M EDTA into a gRNA in-vitro transcription system to terminate the reaction, adding 2.5ul of 4M LiCl and 75ul of precooled absolute ethyl alcohol to carry out precipitation, centrifuging and collecting a precipitate; adding 1mL of precooled 75% ethanol to clean the precipitate, centrifuging, collecting the precipitate, and removing the ethanol; 50ul of nucleic-free water was added to dissolve the gRNA precipitate, and the concentration was measured by Nanodrop and stored at-80 ℃.

5. The system according to claim 4, wherein the precipitation is in particular a precipitation at-20 ℃ for 16h or a precipitation at-80 ℃ for 2 h.

6. The system according to claim 1, wherein the step (5) is specifically: cas9mRNA with the concentration of 600ng/ul and two gRNAs with the concentrations of 120ng/ul are mixed uniformly according to the volume of 2:1:1, and are injected into the embryo of the Pelteobagrus fulvidraco in the first cell stage in a micro-injection mode, wherein the injection dosage is 1 nL.

7. The system according to claim 1, characterized in that said step (6) is in particular: collecting 20 tail of the film-removed fry, and extracting genome DNA; amplifying a target site and a nearby sequence by PCR, directly sending a part of PCR products to Sanger for sequencing, and preliminarily detecting the gene editing effect according to a sequencing peak diagram; recovering and purifying the residual PCR product, connecting the PCR product with a PMD-18T vector, selecting positive single clone, sending the positive single clone to Sanger for sequencing, determining a specific mutation type according to a sequencing result, and predicting the change of an amino acid sequence; PCR amplifying the mutant and wild type genome with the pair of primers dmrt 1E 1F and dmrt 1E 3R, agarose gel electrophoresis detecting whether long fragment deletion mutation occurs; and calculating the gene editing efficiency.

8. The system of claim 7, wherein the gene editing efficiency calculation formula is: gene editing efficiency ═ 1- (1-a) × (1-b), where a is the mutation rate at target site 1 and b is the mutation rate at target site 2.

9. The CRISPR/Cas9 system of the double gRNA site knockout dmrt1 gene in the pelteobagrus fulvidraco as claimed in claim 1 is applied to research on the effect of the dmrt1 gene on sex determination and differentiation of pelteobagrus fulvidraco and molecular module breeding of high-quality parents of the pelteobagrus fulvidraco.

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