CN117947033A - Efficient silver carp bmp6 gene knockout method combining 3 gRNAs and application - Google Patents

Abstract

The application relates to the technical field of silver carp (Hypophthalmichthys molitrix), in particular to a method for knocking out a bmp6 gene at 3 gRNA sites in silver carp and application thereof. The nucleotide sequence of the gRNA is shown as SEQ ID NO. 1-3. The application synthesizes gRNA of three target genes through in vitro transcription, then mixes with Cas9 protein, introduces the mixture into animal poles of single cell stage of chub fertilized eggs by microinjection, and obtains chub F ₀ generation of target gene mutation after hatching. The application realizes the gene knockout of the silver carp with high efficiency, and provides an effective technical means for developing the breeding of the silver carp and the research of gene functions.

Description

Efficient silver carp bmp6 gene knockout method combining 3 gRNAs and application

Technical Field

The application relates to the technical field of silver carp (Hypophthalmichthys molitrix), in particular to a high-efficiency silver carp bmp6 gene knockout method combining 3 gRNAs and application thereof.

Background

Silver carp (Hypophthalmichthys molitrix) is an important freshwater aquaculture fish in China, and is one of the unique four-family fish in China. The 2022 cultivation yield reaches 387.98 ten thousand tons, and the second freshwater cultivation yield is realized for a long time, so that important contribution is made to guaranteeing stable and effective supply of aquatic products. The silver carp belongs to typical grain-saving farmed fish, mainly takes phytoplankton as food, does not need bait feeding in production, and saves resources; silver carp can also control water eutrophication, regulate water quality and has great ecological significance. In addition, the silver carp is low in price, high-quality protein can be obtained at a lower price, and an important effect is played for improving the life quality of vast people.

The gene editing technique is a technique capable of precisely modifying a biological genome by inserting, knocking out, modifying or replacing a specific DNA fragment, etc., realizing gene site-directed knocking out and exogenous gene site-directed integration, and editing a target gene in an organism (Wei Jingliang, wu Tianwen, ruan Jinxue, et al genome editing technique improves the research progress of livestock [ J ]. Chinese agricultural science and technology guide, 2014 (1): 7.doi: 10.13304/j.nykjdb.2014.003.). In recent years, three genome editing technologies of zinc finger nucleases (Zinc finger nuclease, ZFN), transcription activation-like effector nucleases (Transcription activator-like effector nucleases, TALENs) and clustered regularly interspaced short palindromic repeats-related protein system 9 (Clustered regularly interspersed shortpalindromic repeats-CRISPR associatedprotein9, CRISPR-Cas 9) can cut DNA double chains at target sites, induce cell endogenous repair mechanisms and generate homologous recombination repair or non-homologous end joining, so that the knockout, site-specific integration and replacement efficiency of genes is improved (Wang Wei, hu Gongxia. Research of gene editing technology in fish and application prospect in aquaculture [ J ]. Chinese aquatic products, 2019 (09): 87-88).

The current gene editing technology is widely used in the aspects of economic fish character improvement and gene function research (SunY,Zheng G D,Nissa M,etal.Disruption ofmstna andmstnb gene through CRISPR/Cas9 leads to elevated muscle mass inblunt snoutbream(Megalobrama amblycephala)[J].Aquaculture,2020,528:735597.DOI:10.1016/j.aquaculture.2020.735597).

A plurality of SOCS mutant lines were successfully established by using CRISPR/Cas9 system, and compared with the contemporaneous Wild Type (WT), the SOCS a mutant heterozygotes (SOCS 1 a+/-) and SOCS b mutant heterozygotes (SOCS 1 b+/-) have the advantages of enhanced growth performance and obviously increased quality. However, researches on gene functions, genetic improvement breeding and the like of silver carp are slow, and are always limited by the lack of effective gene editing methods, the traditional single target site mutation has low targeting efficiency, and nonsense mutation is easy to cause.

Disclosure of Invention

Aiming at the defects of low targeting efficiency and high uncertainty of DNA autonomous repair and easy nonsense mutation generation in the traditional single-target knockout, the application provides a method for knocking out the bmp6 gene at three gRNA sites of silver carp and application thereof; according to the application, a silver carp bmp6 gene (the total length of the gene is 54028bp, the gene comprises 7 exons and 6 introns), three target sites are designed and synthesized on a first exon of a gene structural domain, then gRNA of the three target genes is synthesized through in vitro transcription, then the gRNA is mixed with Cas9 protein, the mixture is introduced into an animal pole of a single cell stage of a silver carp fertilized egg in a microinjection mode, and then the silver carp F ₀ generation with target gene mutation can be obtained after hatching. The application realizes the gene knockout of the silver carp with high efficiency, and provides an effective technical means for developing the breeding of the silver carp and the research of gene functions.

In a first aspect, the examples disclose gRNA targeting the silver carp (Hypophthalmichthys molitrix) bmp6 gene, the nucleotide sequence of which is shown in at least one of SEQ ID NO. 1-3. Wherein, the bmp6 gene is shown as SEQ ID NO. 8.

In an embodiment of the first aspect, the gRNA is the bmp6 gene first exon.

In a second aspect, embodiments disclose knockout compositions of the silver carp (Hypophthalmichthys molitrix) bmp6 gene, comprising the gRNA of the first aspect and a Cas9 protein.

In a third aspect, embodiments disclose a knockout kit of the silver carp (Hypophthalmichthys molitrix) bmp6 gene, comprising the gRNA of the first aspect and a Cas9 protein.

In a fourth aspect, embodiments disclose a method of knocking out a silver carp (Hypophthalmichthys molitrix) bmp6 gene comprising annotating a first gRNA as set forth in SEQ ID No.1, a second gRNA as set forth in SEQ ID No.2, a third gRNA as set forth in SEQ ID No.3, and a Cas9 protein into fertilized eggs of the silver carp single cell stage.

In an embodiment of the fourth aspect, the annotated mass ratio of the first gRNA, the second gRNA, and the third gRNA is 1:1:1.

In an embodiment of the fourth aspect, mutants are selected from offspring of the silver carp.

In a fifth aspect, embodiments disclose a method of making a gRNA of the first aspect. The preparation method comprises the following steps: designing and synthesizing primer pairs of gRNA; carrying out PCR amplification on the primer pair; recovering the gRNA from the PCR amplified product.

In an embodiment of the fifth aspect, the primer pair for obtaining the first gRNA as set forth in SEQ ID No.1 includes an upstream primer as set forth in SEQ ID No.4 and a downstream primer as set forth in SEQ ID No. 7; the primer pair for obtaining the second gRNA as shown in SEQ ID NO.2 comprises an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO. 7; the primer pair for obtaining the third gRNA described by SEQ ID NO.3 comprises an upstream primer described by SEQ ID NO.6 and a downstream primer described by SEQ ID NO. 7.

In a sixth aspect, the embodiments disclose the use of the gRNA of the first aspect in breeding a mutant strain of silver carp.

The gRNA, the gene knockout composition, the kit, the method and the application provided by the embodiment design three gRNA target sites aiming at the first exon of the silver carp bmp6 gene, the knockout efficiency is obviously improved, and the F ₀ generation knockout efficiency is up to 86.11 percent.

The screening method provided by the embodiment can be used for obtaining the silver carp gene mutant strain. The method is simple and easy to operate. The silver carp gene mutant strain is obtained by using a gene mutation method, and has important scientific significance for developing gene function research of aquatic organisms, revealing genetic development rules of the silver carp gene mutant strain, developing and utilizing genetic resources to cultivate high-yield and low-oxygen-resistant varieties and the like.

Drawings

FIG. 1 is a schematic diagram of the locations of the Target sites of the bmp6 gene provided in the example, and Target1, target2, target3 are the gRNA sequences of three Target sites on the bmp6 exon 1, respectively.

FIG. 2 is a sequence diagram of PCR products of the mutation site bmp6 gene of the parent F ₀ generation cultivated by silver carp provided with the mutation in the example. In the figure, the box represents the target sequence of the bmp6 gene, and the triangle represents the mutation position of runx2 b.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

1. Experimental materials

Wild type female and male silver carp parents are fed into a Yao Bay test factory of a Yangtze river water production research institute of China aquatic science research institute. Embryos used for microinjection of silver carps are obtained by semi-artificial propagation and spawning of sexually mature male and female parents.

2. Design of gRNA (gRNA)

The bmp6 gene sequence:

caaataaaaaaaaaacaaataacgggtacggtgccagcttgcatgcctgcaggtcgacgataagcagtggtatcaacgcagagtacggggactctacaaacatactctgtggggatcagcctagggttaaatcaaagaactgaacggaaaatacatatagtaatatatagtaagaaaaataaatgaataaataggctactgaattggaagtgacgaacaacttattcctttattcctcgttttgagttatacacagcaacttaagatttgaggggacatacagaagtttccagctgtcttctgtccggagcgaagtatcacgccgaagaattgatggagattttgcgaattgaaaaattccctggacaactgcatttgctttgcacgggataatacagttttacagggacaggttacagttacgacccaaagggggttcacaacctgattccacaggagaacctagtcaataactttattctgtagttcttcagaaaacatctactggtactggtgctttaaagaacccagaaaccaacacactgatctgaagatcctataatgtaacatcaagaactttttaatctccaggagatcatacaggcaaaatggttcttaatgagaagaaggttctttgctgaacctaagaaccattgaagaacctggtaagatctatggcgtgaagcagaagagaagaggacagttttccaagtgattatatttagaagtgattaca

ccctcatcatcatcaccaccacagactggatgtaaagatgacaaacgtctggtttgctctgctgggcttcttgtggagctcttctttggcaggtagtagttctgtactggacggttctgagcttca

gcccaacttcatacatcggaggttacggaatcaggagaagagagagatgcagaaagagattctgtccatactgggactgaaccacagacccagaccgcatctgaacagcggcaaatat

aattcagcacccctcttcatgctggatctatacaacagcatgtccactgaagagaagagcgacgtggatcagtacagatcactgtttaccaccacacgacccgttttagcttcagttcatgac

accgagttcctgcacgacgcggacatggtcatgagttttgtcaatttgggtaagattaacttttatatacaacttcacgcttgtgtccattttgttagagacgatatataagagtatacgggtcgca

ctacaaacccaatcagcgtctgtgaagtgaattaattgtttacagattaattttatatatatatatagagagagagagagagttgacttggaaaaagtgtcccaatcaacccgaattcataacaat

gttattttaataatatattttatacatttataaaaatttgggcacaaatactccaaattacctaaaaaaatttattaatatattaataaataatgttttaatcatctaactgctcgatttctgacttaaaggtg

cttcagttctgtttagattaagatggcacaagcacttttcaggcaaaaagagagggttgtaaggtttcaaatgataaaacaataaatgtattgattattatatgagctacacctgtggttattctgct

tggacacctgtgtgaactgactttataaatccactcataataaatcgttcataatggttcaaaaagtgtattttaagtgtatttctagtgcaatgacattcagccatgtaagtgaagttagtgaacaa

atatatttgagcaggatatttacatttttaaacatttaaagctggtcaaacttatttttgtaaaacatttagcttgaacatgttcttctgctgtttttgtatttgatactttgttacctaatgcaatgatttaca

cattttaagtgtgaaataagttttttgtggccactgcatttttaaaacctttgcatgcaaatacattttataaaataaattgtttaatgaatctaaacatgatagaattcctcttattagtgaaccaatgtg

caggccatcattgcaaaatgtttatttttattatgcctcctggagtgaaaatgtttgattgtttactgtagcttgcaggattaaggcagtttaattttaagtaatttttaattaaagtgcacattcaagca

ttttgatattttgctcatttaccatggtaagtgaagtaccttggagtactatcatgttcatttgtaccagatgaatcatattgtcgcctcagtagttttttgtaagattctgatggatatcacagttcaac

actttatttactgacatctatcaagttcatggtgacccagagcacaatgtcagaccacaagtctgtttatgctcagggattctgtgtggtttgcttcagtaaacacagacctcattaatgaaggga

gtatttgtgtccaagattcacttgtttatgtgaacttccctctacaggggccaggagttgcccaattaagttaattataacaataaaaaactcttctggctctactcattgcagctatgggccaattg

ttgggtcagttgccagagatgcttttgtgtgagttgcgccattctagttgttgtttgggttcatttcagagctcatttctgtctgttttataagacgataaatgtgatctggtagtggttttctgcagtca

gttgaacacttgatggaaacatgtcattgactgacatgccacattttcagtgctgcttggaaataagcagaatatcacatcagctgaccacagtgtgttacccgagaggcttgaagtggcttttc

acatttgctgtgtgtcagtgactcaatttggtcagttattggatattttgcattagtaattttaatatggttttgttctagactcgtgtaaggtatgtcatatcatttgttttggatgacttgcctatgattca

agtgttatttcttctctggctatttactgcagtcatctgtctagaacacctactagtctccgaatataggagaaaaaggaagcactggtatatactggcatacagaaactgactagtgttgcaact

catccagaaatagctaggtttgtcacagacatttggatagtcaatgtatcctatgtgaatcatcttatgactgtagaaacagtagtgcagtagtgttttccatgtagttcaattttaggctcagtgttt

acactctttttttatacactgaaagcatacaatggctgtcaaactccgaaaatttgaaaaacaaaacaaaaaaacgcatcataaatgttgtccatatgacttgtgcactatatagcctattgctttat

ctctatatctgttttttttttttttttttaatgctgtacaatagctttgtgtaaggaacagaccaaaaatgaaatctttattagaatatatgcaagcagcgcctaaaattaacacttgccaagcaccaaaa

gcgagtcaaaataggtgttggtgagtatatgaaacggtgtcactcaccagttggccggtaacatttttataaattctaacaatgcatgaatgtgaacaaacgtgaacaacgctcagaacagtt

gacgggccagtgctacattgtcacgaaagtttaagcgaatactaaactaagctctctttcatgtcttcttgcacttaagcagacaaattcacacaaaattatgttaaaaaaaaaagaaaatagtg

agtatcttagtaatcatagtcgttatgtcttaaatgaacgtaaacagttgaaaaagacgcatgtgtacaacatcagtatattatatccgtgcatgcgctcttaaagtgacagcagcctaatatttcc

gctgctgtctgtgtttttaatgttaatcaaacaacaaaggacaaagaaaaaaaaaaaaaaaaaacctaaggattaattaataaggatcagacctacctaaaaaaaatacctgaaaaacactgtt

tatttcatttgtattattgttatattgtatttatttgtgcttttgcttgtaatttgattatttgttctcattttattactgactgtttactgtctttaataatgttttacattatattagttaggttagtagatttagctgt

acatttacatttatgcatttggcagacgcttttatccaaagcgacttacattgcattatactatacatttgtatctgagtatgtgcaatccctgggatcgaacccacgaccttggcattgctagtgcc

atgctctaaccactgagctacaggaaagcagaaaagctgtggtatacaaaattgggatagagaattgtgaattgtgaattttcgctggtatctaaaatgttggtgtcattactgcctttcatgacc

ggtaaaaaatatttatggccagtaaattttcttaagtcaccggccattggcaggtgtggcaaaaagatagtttaggcacatttgtgaccctggaccacaatattgagaaaattgcctataaagat

tttaaaattattatcaagtactttacatgtgctttacaatgttagtcaacgcattaatcattaaaaaagatgatgacatcactgttttctccagagctgctttatgaattgaactcattctacaattgatg

aactctacacaattattgaaccaaactgaatcaacactgaactgatttcaactgaataatgactgtttactatgtctttttagagctgctttagagcagagtttaattgtgtttgtgttaattgtattcaa

tttaatcattgaatctttattttccttgaaacaatctgtaaataaaggtgactttacgtgacgacatgaaagcatgctacatgcagatgtccactgaagtcgaactttgagtttgacagaattgcgttt

gagtgatttttgtgacagatttaggaacatccaatgttcttgatgtcaatcccaatttttccagaccagtcttgctctgtttacagaagggtagaggataccctaagcttttcttggcacagagaat

gttgaggaaactcccatcttttgtttgcactagtaatattgtgtgtttgtcttttttttctggataaggggggatctgccagtaatccagggaaaatccatggggggtggggggatgagcatttctg

ttttctcttctttggtccatcattgctttattccacctagatcttttgtgttcactcacatttcctcctataactccctcattacaaacacatacacatttcaaactcattcttttccacggcacacgtcttaa

ctagagcccagctgtcatttctccacttagcaaagtcaacagccacccaggagagcacaggtaagcttcgctttagacctgtaaagtagtaaaagtgacccacacgtagagttttagggggt

ttcttgggagcttaacaaggtctacaatcctggcttcttataactgtatgaaccctgtatttcacaatgcactttcatttcgcttttcgcgctttataacgtctttcagtgtcctgagctccaaatctgg

gctgagctgtttgttttgcaccacaatgtgtttctaaacccacattttatcacataattagtttgccacaatcaatcaatgaattgtttgatgttggcaggttaggcacaaaatggatgaccccttttg

aaagactgcccatggctcacagacacaggagggctgtatcacttcgagtctgcatgcgggcgttaacatggaaattaaggaaggaatttgagttatgctttagctcagtaaagaaggaaag

ggatttctcatgggtgactcagcagactgataagaactgttgaaaacacgcagcaaatgagactaatggccaggcacagatgaaatctgcagacatttttttgcactgattttgagggaaaat

cgtctttgggatggttttaatgtagtaaaatatgttatatggaactgagagtcattaacaaatttacatgtagtcatttagctaaagctttcatccaaagtgactcaaaaatggtagctgagaacatt

atcaaatgatcaagtcatcatgaatttgacactgactttttttactttctcagtgcatgtacttggtcttcatgtgcacagtctattggtgaatgttatcctacccatcaagataacttgctttgaaaaac

gttcttagctttctaattgttttctgtcatatggagaccaccaccatctaatcaattccccagagatgaaatccctctatacggttccctttcaagggaactcacgctgcgtccaaagcgctgtgg

gaacatgtctgcttgattgcgtcgtgatgcacatgtgaaatcagtccaatggcgtgatggaacgtcataggcgggtgacgtcatgaccaggaaactataaagcactcctgaaccaaacagt

gttaccttccgtgccttcaacaagcgctgtgtgtgaaattgtctctgtctaaaaaaaaaaaaaaaaaaagtgaatgttaccagctgagctcccccgcagagagactggggctcgggtgggc

actcacagccgaagtcttctcttgataggccggatctgagggccgtccttattgctgagaaggctttggcaaagaagccctgatgccagatgtgctgtgcttctgagggcagtcccctccgg

ggcgaatcagtgttcacctcattacacggtgcccgtctccccttggtgctctcagggagctgttctgccaaccctgccacctcaagtgcttcagggcgcagcggttctcagcgaacatatgt

ctcattgtccgcccagaaacgtagcggctctaggacgttcgctccctctgaggagggtttcagaggggccagttcggttgttctctgccagttcgccgcttcagggcaccgagttggccact

tgtcttacaccagagaccagtcttgagagactggttccttttttttttttttttttggcagtgtggaaacttctattaaatatatcttaatgggttctgcagacagtagaaaaagggtacagaattcagt

tcgggtttcgcccgcccaggttcagaagggatttgtggtaaaccctgagcaggctctggtcatggaacaggaggtacaggctccgctctacagggaaccactctattgcctgcagaatga

ggcaatagagcggttccctcctcccagcagggagtcgggttttacagccagtacttcatagtttcaaagaaggatggagggttgtgtcctattttagatctaaggcagttatatcattgtcaag

aagctcaatttcaaattgctcactttgagacaaatcatgtaacaaatcaggtccgaagactggtttgtcactatagatctactgtaaaagatgcatagctccatattcccatcctatcgggggcg

aagcgtaccagtattgggttcttccgttcggtctagctttatctccccgcacattcacaaaatgcgtggatgcagctctggctcctttgtgactccagggcattcgagtgttgaactatatcgaca

actggctcattctggcacagtcagaacagttagcagttcagcatcgagatgctgttctcgctcacatgaaaatgttggggttaaggctaaatgccaagaagagtgttttatctcaggtacaga

gaacaacttttctaggcgtagtatgggattcaactacgatgcaggcacatctatctcctgctagagtagatttgattctcatagccgtaaacaagttaaagctaggccaatcactcactgtaaaa

cagtttcaaagactgttgggtctgatggcagctgcgtccaacgggataccttttggcctgctgtacataagacccttacagtggtggctcaaaatcaaggggttctccccgaggggaaaccc

atttcgcatgatcaggatcactctgcaatggctttatgcctgtgtcatgtggaaaaaacatttgtttctttcccaagggccagtgttgggaactccttgtcgttgcatatgcttacgatggatgcgt

ccctcactggcttggggggcgatcatgattggtcgctcagctcagggtttgtggcggggccatcatctttcgtggcatataaaccagctggagatgatggctgtgttcttggcactcaagca

cttcctccaagacctgaggggccaccatgtgttggtccgcatggacaatacagcggtggtctcatatataaaccgtcaagggggtctgcccattatgccgattggcacaccagatcctcttg

tgggcccaaggggaactgttgtcattaagagcagttcatcccggggcgtttgaaccagggagcagacatcctgttgaggcaggggctgaggcctggggagtagagacttcaccccgag

gtggtggagctcctgtgggaaaagcggaagtggatctgtttgcgtcttgaaaaactactcactgtccaatgttttactccctcatgtatccaagcccctctggggaccttgtgtctctcctcgac

ggctctcagtgggagattatggaaactgtgggcttgacctctgagggagaccagctcgtaggttcaggtctctcaaccaaggttgtagagaccatactccattccagagctccctccacaa

ggaagctgtatgccctaaagtggaatgttttcactttgtggtgcaggagtcgcatactggacccagctaactgcccagatggttcagtactggagttcttgcaagaccgattctctgcagggtt

atccctgtccactttgaaggtgtatgtggcggccatagctgcttaccacactcctttagatggtacacccatgggtaacttgcttcctccgtggcactctgaaagaccaggagaaactaaatct

gctctgcccagttagggccttagatgcttacgtccacagagctgccctgtggcataaatcagatatgggcattgcattcccacagcactttggatgcagcgcgagttcccttgaaaggaaac

gttttaggttacgtttgtaaccatggttccctgagagggaacaagacgctgcgttgctttgccatactcccagcatgccagtgaccgacttgcttcaacctatcagctaacactgtttggttcag

gagtgctttatagtttcctggtcatgacgtcacccgcctatgacgttccgtcatgccattggactgatttcacatgtacttcacaaagcaatcacgcagaggcgttcccacagctctttgtggctt

cctgtggcctcataagataatgaagtgtccatcatatgcacacttcagaatctcacaggaagaagtaggtcatccggatactttttgcctgctcttttatgcgtactgtaaattcggacatccttct

tttgtcacatgctgtttttagcctattttatagtagggaagtatgcgatttcagttgcagcacttctaaaaaaaaaaaaaaaaagaattaacgttaattattgcataacttttttgaaaaagcaacttta

aatttaaaagtaatgtgtcacccaatacttgaaaaaatgatctgattatgtaactcacgttgcttgtaatgctttaccccatctctgttcgtgggaaatgtaagttttgcgtcctttgtctttgcgtggtt

acattaagtaagagaagaaggtccggatattacagagcatatgcggtgggaggtgtatttgttctgacagcttgtaaccataaatatggggcaacattattaacgcaatggattgttcaaaaaa

aaaaaaaaactctgtaatataactcatatcaacatcagactgcttttcagagatggcgacagctccaagagggtttaaagcccggaacacaccaagctgacggttggccattgggcagttttt

gtttgtcggccgactaggttttctcagtgtgttccgcaccgtcggctgaagttggtccttgacggctttttttcggccaattcgacatattgaatcagcgtcggacctcgtcggtcagtcgggcc

atctgatcagatcattctgattggctgttcagctactgccacctgctggtgcggaaaggcatttcttcttacacagatgctgaatggacatgctacttggccgtcgggtgtcaagcgtcggtttg

gtgtgtcagggcaactttggacccagacgctgccgacgtgagccaactccacagtctgcattcgtcaccactagttcgttggcgtcggcttggtgtgttccgggcttaaaattgacccgaag

atggccttttttttttttgcttgacaggtaagatacccagcaattgttatgtaaactgtcagcgttgtctaacttttgtaatgctatttaatttaagtcattaataatgaatcaaatgaacagtggaagac

atgtacctgctcaacagacattatttatctttttttatgtagtttcttttctttttggagcttttgcatttttgtgcttttgaagttgttagtgactttgataaaatgataccatcatagaatatatccaactcac

gtaattaccaaaacaatgttcctcctcgagtaacttgcggttcgggccagatcttccctactgctgctttaataaacaaacatacaagtgacaagattttgtgcactcaatgagcactccaattct

tcatatatctgcagagaattttttgtgcacagattcggtgtgagcctactaacaactaatactccaattaataatataaaaattgcaaaaattattttcttgttcagtatttttgtcttgtgtttccagtaa

aatatctaaaaatttttaaatcaatatacatttacttgagaagcaaaattacataagatatagtcttgttttctggaaaagccatcaaaattaggttactttatgcttaaaggattagttcactttcagat

gaaaattaccccaagatttactcactctcaagcaatcctaggtgtataggactttcatctttcagatgaacacaattagagttatattaaatatcctgacgcatctaagctttataattgcagtgaat

gggaccaacaagtttaaagctcaagggcatccatccaaagcaaaacgtagtccacatggctccaggggattaataaaggccttctgaagcgaagcgatgtgtttgtgtaagaaaaatatcc

atatttaattcatagttataaagtaaaatattttgcttccgtcagaccaccttccgtatttaacttaggaagaaagcgtaactgacgtcgcgtcagttaagctttttttcgtaagttgattatggaagg

cataggacgtagtgtaagcgttttgaactgcgagaggcgttacactttcttcgcaagttttcatctgaaagaagagtgttcatctgaaagaagaaagtcatgtacacctaggatggcttgagg

gtgaatgaagcttggggtaattttcatttgaaagtgaactaatgctttaaatcagtaacacatatatggtgtaatagaaaataaaaacttgttttttttttatcccattggcagatatttttttcttgtttta

aatataaactcacttagttttgacaattttctagaaaacaagggccagtcacacagaactcattttttccttccactgcaaaattttccattgtttttcaatgttaacacacactagaacagacgtgtt

gaaccattgctcttttgcagcgtcccgtgcatgatgtgacattctaaaaacatggcgctcaagctaaaataagttcaacttttaaaaaactcatctccagaccttgctacttttttttttttgctccact

gacctccacttaaaggttcaatgtgtaaattttaacagcatctagcggtgaggttgcgaattgcaaccaacggctcagtccaccactcacccctccctttcgaagcacatagagaagctacg

gtggccgacacaggacaaagatgtcgtcgtctgagacagcagagagtagccagtcaagcaaacacgctctgtaaagcagtttgttcatttagggctactgtagaaacatggcggcgcaa

aattttacggttacgtaaggtctttatacaccactgaaaacatagttatgtatattatattgcatttctctcaatagatcctcctaaatattacacactggacctttaaaacagcaaaaatgtgttctgt

gggaaccggcccttaatatcttgcatctcaaagtcaatgtctcttttaagaatgtttagatgtctgtactggaaaacaagagaaaaaaaaacaagatgtgatttgttttatcaatcgtaaattgaaa

agtggtcactttgagaggtgtgatgtcagctgaaagaaaaaccatttcaggggcgaaataagttattgcattttgatgtaagattacgaagacaaacaatttttttttcctttagagaaaaacatg

gcgtgataaacgtgggaatgtgttgtaataaagtagcagtttatttatgcaggccaagaatgttgtagttacaatacttgtctttaaaacatacaaaaaagtaagcattttgcagagactttgtcca

catggacctttcttacattcaaaacactggtttcttggttttgtttgttttaatgaacaaaatggcttttgctgttcttctctcttagcctggagccagatcctctcgttctctgaactaaacatttttgga

ctgtgattatctgagaaagaatgcgagttttaatctcagtcaaacagacacgctgaagttgatgatcgtgaggtcaggagataagcttctttcagggaatgtcctctttgctttagtcttagctgt

gaagtcccgatgctatctctgcgtctcacgccatagctgtcagacatgcccccacggccactggttcctgcgatcagtcctgaggaatgtgccgttctctgcaggtagctgctgtctttgact

gaagcatctcgtgttatcagggatcatattgaatgaggaatgaataaggatctcagaacaataaaacttgccttgccgttgcctctggctgtgaatctgtcagataagagggggtttgttaaac

cgatggatagggacgtgaaaagataccacagaaccaacatctgattatgtgataaatcacttgtcggatgaatcacatccatgtagccattcaaaagtgtcacgccaacaaccgaggagtta

accacagaagaaataaggggtcacccctgcttattcctccaaacccatgttaaagccaatgcttgaacgaagtctgtgcttgaaagtgaaacgcttatataaagtcagagatgtgtggacag

cagagtgtgacctccctgccaatccttttgggttaaactttgacccccagatgaggtcacagctgagccttcaccactgggttaagacctacaggtatgcaacatttgttaccatatgatgttat

gtaatagaattataaatatgaaaattctgtcattaattcctcacccttatgtcgttccacacccgtaagacctttgttcaacttcgaaacaaaattaagatatttttgatgaaatccgagaggtatatg

actccatagacagcaatataatcaatgcttttaaggtccagaaaggttctaaagacatcgttaaaacagtcgacgtgactgcagtggttcaaccttaatttaatgaagcgacgagaatactttttt

gtgcgcaaaaacaaaatgaaaataacgactttattcaacaatccctattatgctttttcaaatattaccttttatgcagtgtgtaatgtgacaaatttgcataataccagtttattagtgtttttttacttg

gatgcaagtaaacctattgtaggagacctttaaaacaaattaggaacctttaaagtagcataataggggcattttaaaataatttagttcctgtatttttaaacaaatcaaagcaattctggaagaa

actgatccctcactgacattataaacacacactctccacccatggcggaatgtcacccagtgccctccacaagtgaataggaaatgcgtgagcgctctagatttatggctccattgctttcttc

aggcagcaatgaatttgtgttcatttaatggttagtagcacaatgaagtgtctcgtggtgtgctgaattagtcatctaaagaggttgcattgccagaacttttaataacaatcagagtgcaaaaa

gaaaaaaaaaagaaaaacgaaaagcatattaagcccaaagtatttatgtgtacagttagcatacacacatatcaatgcacaggctttcaaagtatacttcatttgatagctagcgtgaacaaa

ggcactcgatacatacacactagaaagattcatccttgtccagtgtctgcactatttttgtccaaaagttatgagtgataaaaatgtctttgtactcatttaaactcaatttaaacacaagcactcat

cagcagatgtctgttgtttctgccgtctccagtagtttgttgttgttcttgttctgtctcatacttttttttttgactatgaaacaacggcctgggccagtgttgccaccttgacaacataattagtgtca

aaaaattcaaagcgcctatgcgagctgcatataccattaacaagtataccgctagggtccacatacagtgtgtgtgacatgaatttcgtcatttgtactgaacgcatacagcacaatttttggaa

gtatccatatttgtacaactctacaaggcttaaagattataaagatgggttataactaatggcaagagattgccctctcctgtagaccaggagagttaatgtgagttgacttaatgcacatacttc

gaccacctgtgtatgtgtacaggctatgtgtaaaaaaaaaaaaaagcaattctcataattaaactattaaaggcacttgatggtttcatgtctagatcgcacgtacgatcacatcggtgtgatca

ctctaggttggtccctggagcgtaaaatcacaccggtgtgattagaattttcagtgtgtcacatgatgaactgctaaattaaaatcagtgttcagttcaatgttttttaaccatgtaattagggccc

gagcaccgatggtgtgaggaccctattgtaattgctcagtcaattcttcttcttctccgaaattaattgtaattttgagggcctaaacatgctcgaaaactcatgaaactttgcacaagcgtcaga

agtgatatgggtctgatatgggtttcagaattaggcgtggcaaaatggctcaataacgccacctacaaaatttcaattaagcgcccttcgcgctacgtttcatgtacaagtatgaaatttggtac

acacatgtaacagcccaatacctacaaaaaagtccctgggtgcaaaatctgaaaacccaacgagaagtgacattattttgaattttctctgcaaaacgtttggagtttttgccatttccagacgtt

gtgctttaaagaactccctcatagagttttaatcagatcaacatcatatttggtcagtctaatctaaaggcctttgcgacattaaattgcaaagatcttgagttttcattgaagggcgtgtccgtggc

ggcctgacaaaattttgatatttcgccatgaaacaggaagttgttgtaactcaggcataaaatgttcgatctgccccaaacttcacatgttttattagagtcctggcctgaagacgtctacatggc

aacattcagtcagagtcatagcgtcacctgcggccaacaggaaatgacatgttttacactgtgattaactcctcatagagatttaaccagatcaacatcatatttggtcagtctaatcttaaggc

cttaacgacgttaaattgcaaagatcttgagttttcattgaagggcgtgtccgtggcagactgacaaagtttgatgtttcgctatgaacgaggaagctgttgtaactcaggcatacagtgtctgt

gataagagtcctggccggaagacatctacatgccaatattcagttagtcatagcgccacctgctggcaacaggaaattacatgttttgcactgtaattcactcccagaaacacatttcaatatg

ccacctagtaccaaacatgctagaaacatgttaaatcatgcaactcttggctaagtgctaaagtatgtgattaatgccatgaaacaggaagctgttgtaactgtctgatctgctccaaacttctg

ataatagtcctggcctaaagacatcatggcaatattgagttacagtcaaagcgccacctgttggcagcaggaagtgtggcatgtcaaaatgactttgccatatttctcctgtatttactcgcttac

atgcatgtcacccactattcactgttttcctaaggccatcgggtggcggtggccctgggtgctagggtcctttcgtcgctgcttgcagctttaatgtttattttagatttcagacatttaaatacaca

taagtaccagaaaaacaacatttatagtttgtcaaatacacatcgatgttgcactgaacactgaaaattttatgtatttcaggagaaatggaaatgctaaatttttagcattgcagtgcaaatgtct

gcagtggcgtaggccgtagggagtatgacgcatggaagtagctttaggggcctaataattagagagtcagacttgtaacccgaaggtcgtgggttcgggtcccagtactggcaggaaat

gtaggtggggggagtgaatgaacagcgctctctcatcgagcaaaccacctaacccccaatcactccccaggtgccacagcaaaaatggctgcccactgctccgggtgtgtgttcacgat

gtgtgtgtgtgctcactgctgtgtgtgtgcacttggatgggtgaaatgcagagcacaaattccgagaatgggacaccatatttggctacaatgtcactttcaaactaacatggtggtacccatc

acccattacagatcaatgttttatttcgagatatagttaacaagtttgtgcatattttgaataaagtaaaaataaattaaaactgacacatcggttatacagcactattttggctgctgtgtgtcacgt

gacaagcttgatgcatcacaattgaaataatacagtgatacattctaaaataatggttgccttaaaaatttggggggcttagcatattttaaacttagttgtgatgtgtgaaagggttaattgagca

tgagatcggttattggttacacttagaatatgtcctacctagagtttccacgaggaagaaatctatggaggtggatgtcagaggatacacacagcgacaaagagagagagagagagagag

agagaaagagagagagattggctgttgtacttgctgctctggaatgcaagccaccaatattttgacttctcctttggaaacagctgcgtttcctttttcaaagatattttttccttatttatcagatcc

cttaggggagatccatctctgctgtctgacacagacttcctcctctaaatctgtgtctctggccagccctgagctcacacacacctgctgcagcacacacagatggctctaacaggtctctctg

cttggcatgctcacacagacacagacacacaggtagccactgttgtaggagacaccgcacacaatacctcaatacacaccactggtatatctcatctgtcccaagtatatgtttaaaaattag

gcattccgacccccttactgacaaagaaaatcgctacctagggtcttggggttactgtaggtttccctaacactaacctctcaatttgtgttgagcagtataccagtttaaattaaaaactggtac

aaaatattcaatttgttgatttttttttttttgtataccattctttgtaagttaaagtgactgtcataaaatgcagtctaatcttgctacactgcaggatgtacttgatcttataggggctatatgtaaaattc

agaaacccttgttataagcgacaccagtggctgttaaataaactgcagccagcaacttattgctcgtgctcacacttgtgcacatagacgcacaagacaccgaatgtgattaaatgccatgat

atactcacgccaaagtttgttttcatttgtcgcgaacaaaaagaagttttagaagagatatactgttaacgaacatccttatgctgtccatgctactgagagctacgtttagatgaaaatgacagc

ggtgagaaaacagcagcatctgaagtggatatgtttgcacaagctctgcaattcaccggcatcattaaattattgcagggcaaatttattataatattattttagtaataaaagtaacctaatagtc

ataataatagactagaagaaagaaacagacctacatcatgtcgactgttatgatagtttgattataaagtatactgtatttgagactatagactatatctggctatttgagcagtggcgtagcgtat

tctctcagagacgacgagagactaatttcaacatgatatgctgataacagtataaaactgtctttatttattccattatttctcttttggcccgtacatatagtgaaacaaatgggaaaaatagtattt

cgtgttaaacagttggtgcttgaagtaaagctgctcttaattttcattgatgactgcaatgttaggaaatattatagactgttaataattttataggtttataattcattgtataatagacctaaaaaaaa

gttttaaaacattttcaaagaggagctaatagcctaatcttttattatcctcacagcatgtcagttatgcggtgatgcgctatgaaattattggagggcaaatttattataatattattttaataataaa

agtaacctaattgtaataataatagactagaagaaagtaacataagacctacataattggaaatgccaaatcctcttaatattgccagtatttgatgcttttgccaatatctcaggctattgtccata

catgatcatcgtctattgatgcacagggaggttatcgcggggtccctccaacacccatagtgtgtgccctgcgtgcccgtcctttacgccactgtatgtgagactatagtttgattttttttttttttt

gcatgacacattgacattacagtacagaattatcctgaacattgaagtattcatttcatgtgtaatagaagagaagatctcggaagcgctcgtaatcctcacattctttggattttcccggcaaaa

ccagcccgagtccttcacataacaatcagtctataggcttaggcaaccttggaagtcggggatggtttcattttttaagttttgttacaagctgtttacacattggcaatacaaaaaatgttacaaa

accctatgacaaacgattaatacatgaaaatgcttacatatatatatatatatatatccccctttaaaaagatttatgaaagggcacctactgtaaaatgcttttttaaaaatgtacctttcctttagtat

ttagatgtttgttcatgtaaacaatcttaaaagttacaaagcacaaagtccatgccagagggacttaaaagggacttaatttctccaacaggaaacactagcctgaaacgccttgtttgtagtcc

tgccattatttctgtgacttagcaacgtcacacatttgcattagcccgccctcaaagaacagttggctaagtgattttgctgtgttgaggagactgattatcgtacaatgccaaaagtgattcattg

acagtaaagctataatgttattggtttacaaggcatgcgtgatgcaagttgaccattgcgctttgtccaatggtagagccacagaggttggcatctcctattactcacgtttttttacctgccaggt

gataaaaccagtactgcaattaaaaaatatatatgtattgattcaataaatctgtcaaacattacagctgcatattaatatatatatatatatatgtatgtatgattacttgtgttgcatttacttccctaa

ctggaaactggctatctaaaactaaaaattggacatattttgtttatgtttttataacattatttatcttcaagaaaataagtccaagcatatttgttgtgtcatttttagactttgagtgttgaggcctgc

gctctagtaatgctatttggatatctccttttgtgaggttgccaaagcacagactaccattagtctatcccaaaatgttttgtcagctgttctcatggattatgcctttattaatgtttctatatgccgaa

aagaaggaaagaatgggactgtttattttccagaccatttcctccattttatggaatttcctccttatacaaaaagacattataatatttgttgacaggtcgttggcttgtgtcggcacttaagtgaa

aatggatttgttttatgcctgttaaagacagaacagctgactcttgtaatgtctgttagactgttgccctacacatatgcagaacataaagtaaacagttctcctacacaaagcaagttaacctga

ctttgtgcctgaactgttgaatacatacacacacatacacacacacaattcctctctgttctgtataattctcctactgacatgcaggtatggccatctgactgcggccattcactatggagacga

tccgcttttctgatgtctcacagtgtcttgagagatcttttaacagtcataattattgcaaattactgctgttctctaaataccctgcaactacagagcaatgcagacagatttcatttcttcctgcatg

aaacttcagatcagtccaatgaagatttgatccaacatactataaaaatacattttcctcaaggtcactaccgcaaaataaaactgacagggtgatttaatgcaaaataaacctgacactccctg

acttgaagcagagaatatgttacaaggcaacttgccaaataaaaaaatgaacctgaaatattgatttgagtgtaaattattttattatgtgagaataaagagagtgtgaagtgatatgagagaca

tgaaactagtgcagctatacaggaaataacttgcttggaacaacagtcattgaccaatcaagtattcctgagagcctttttatgatcttaatgaactattatcagttagttgaggccgatttgcaat

atatgaaatatattttatctttatgttccttcataaaccacttacagcaacttgatgtatttcatcattaggattttcttaatggaagaacattattacttttaacataaaatatagtaagtttaaaacattgt

acactgtaaaaaaaaaaaaaaaaaatctgttaaatttatggtaaaaaaataattaactgatataatgttaatttaccaacctattgaagtactaatatgtaagtaccatcatggtaacacacatgaa

attttaaaaatgcaataaacattaatttaacattagatgtaacataaaaccctaatacacataactgattaaaaaaaaactatgaagaaactatgaagttatttcaatgaaaatacttcaaatgtgaa

gtgtcacgcagggaattttggaaatgtcaatttatggtttttcactgtaaattatacaatgacttgttatttttcacttccaaatcctgtaaatttaacggtattttaccgtaaaattacattaaatccacc

aatagatctattacagttatttaccatatatagtacggaaactttctgtaaaccaattaactgtttttgagtgtgagctttttcctagtgctcacatcaaccaatggatctttatgaccttctaaccatct

atggtttgccataaactactgcacctcttgctgacctctgctcagagctctgactcctgtagtggcgcctttgatgctttgcgtgggtcaaagacctagatcagagttgtaggaaatgcccagg

gaatgctatgtcaaacccctgtcatctcccagctgcccgagtacaaaaccctatgacaagcaggacttctgggcggccaagaggctctctagtgaccgtctgcagtctttagtctccttgtta

gagcggttcgaaccggaggggctacagtggtggcccatacagggattgaacccgcaatcttggcgctatcagcaccgatattacccctcaggtcctacttgccacgctctgtgcggacat

agtatgttacacatagtatggaaaaaatactgtggaagtcaactgggcccatcaaatgtttggttaccaatattcttaaacatatcttagtttgtgtacagcagaagaaataaattcatacaggttt

ggatcaagtaatgcattcagtcatgtgacaggatgtgacatcattcagacacctgcaaaggaccacatggttgtgaagcaaagttactaactctctctaaaaagggatagttcacccaaaaat

gaaaattctgtcatcatttactcaccctaaagttgttccaaacctgtatacatttatttgttctgctggacacaaaggaagatattttgtaaccagtttgtaaccaggccactttgaggcaccattga

cttccatagtaggaaaaaaaaaaaactgttcagactgttcagtttcccagaactgttcagtcccagaactgttcagtttcccacattcttcaaaatatcttcctttgtgttcaacagaacaaagaaa

tgtatacaggtttggaacaaccagagggtgagtaaatgatgacagaattttcatttttgggtgaactatgaacaatataaagggaccaaaatggtccctttatattggttactccaaatgacatca

atgaaattctccagacattctttctcataacaaagcaacaatttctacacataattttaataccattttgcactatgttgatatatgatgttataaaatcatgccagaataaaaaaatatatacatttatt

acattttaagatattttaatcactaatgaaatggctgtattggcctttggacggttaaaccgaatgactttttgagagttcaaactctttaaaatactctcatgtagcaaaatataatttaaaccttttgg

attcaataaaagtgatctagttgtactaccttacatactttggatgtcatatctttgttttttattattattaaggcctttggacaaaaaaaaaaaaaaaaaaaaaaagaccccaagatcattgaccat

cattgaatgtaattaccaattacattggcatttcgaaaatgaaatgtcccgtgagtggcactaaaaggttaatgctaaaagtctttattaatctagtatgcccctgtaatcataatatgtgtgaaaat

gaataaaagttattttgcaaatatacgtttttagagatttgcaaaaatataggtgaaggcatgtttttccaacgagcctatgttggaaatgttacaacttcccatcccaacaagttgccagaactga

tttactggtagttctcaacagctcctgttcacttataatctctaaatggtaaaaagccagaatttttcttcaaaaggccatatgtgtaaaagtctgtctgcctgtctatccatggaaatgacaccaca

taaatccactttggtgggcttaagtcttcccaaaacaaaagaaaaatgaaaggaaagcaaagaacgtcactgagatttttaacttaatttcaattcccctcagaagatgttatgagaatttaattc

atgagacaagaatttgtgatggttttctgttgctaaataacttgtcattaaattgtagcacataatttcatactgtataactgcgttaactctgttctggaataggctttataaccgagggttataagc

agttacattttttttgctgggtttggttgagtgctataaagtgtcattcacgtttttaagagtgtcaaaaacaagtgcagtcgtacagatgacaagcaagtcgcagcagcttttcattacagaagtt

gaaacaattagattgtgggggaagagaatgtgaacggggattctgaagataattacataaacacagatggattcttcacatgcctggcttcacagcacatttttattggttaaactgaaggaga

cagctttcaaatcatctgaatgtttcatgtgctgcagagacgcaggaaataattgcaggctttgtgtacactcaacaatttttttttctcacaaatttgtggtgactgattttggattggactgaataa

ttaaaaagcttaagatataaatgatgccttaatccttaacagaaacttgttggtagtgacttgttttgcacccaagtactgcatgtataaagtggatgattatatacattatacagtttattgtatgcat

taaaaatcacatttgttaagatcagtatgattattttaagcgatcacattaacacctctattattatcagaatacagtttataataataaacagactttaatgggtcatttcaattttccatctccacattc

atttaaattggtataggctattaaaatgatataatttcaatacatggaactatcctgtaacattgtcactgtatttttatgtaaaattctggccacctgagctagcggtttgagaaatttattcaaatcgt

tcagccagctttaaacttacacagtcaaaattgtctatagcatctcctggatacttcatagagcaaagagtctggaatataggtcaaaattatttgtaggattgacatcagtgtagtgccccctttg

gtcttggaggaagtgaattgttgtttttcttagtggttgtgggcagggaggaaaaagtcaagatgtggtgagtgaatcaaatgcaaatgaccaccggactacttcaggggatgtgatgaaaa

acatttaaagtgcgactcgcatgtgaccacggaccacactgaagcctgactgcccagtccatgcagccgtatgccactgtgattgggaaaaaagaaaatatctgtctgtctctctctctctctc

tctctctctcttcaggtgtctgtttctctgtctcacttctgcgtgttttgaagtttttccatatcttctctgtaggtcacagcccactgctttttgtcctgtgtgggctaatgacaatagcacagaagaaa

agaaacagatgttatgcaggtggaaatggagaatcagccttaattagaacaacaccgatctttatcccagacccctttatccgttttacaccgtaacaaatattagtacatataaatacattatta

actattcaaatttttttttaaatttattcaactttaactgtatattggcatgttttaagttaaatttgttctattcagaggtgtgtttcccataaaacaacctaactcactgcttaaccaccatagtacaatgc

atcatttgaaaaatgaactagctagtcatgactgttttaggttggttcaacaatacagtacatgactgtcgttaatgatgccatgcgcagatggtggagtaataacttattatgtttattaatcatttt

gagattgaaattaatgtcagattattttagttaatttacacaaacaacagaaagcccttctttttttctttttgttttttggtcaagttaacatgtagtaagtaaattggagttattttacacctgtatgcat

gcggttcattcagtcagccagcagctttctccacaatgtaatttctcagagatactgcagtaatgaacatggcatctgaggactacttcgctgtttttttttcttttttttttttctatgcgtgtaatgata

gacttgcgaccatagttggctaacaatgcttttggaaaatgtacccctgtttagttaagggttattgtattattttagcatgttgtgttggtgacaacaccctgatgtgttggtgtgggtttccaactt

caatcccttctatttttccatgtgtattgtggtctgttatgaagatacaatgctgtttgtgtaattttttagcattgtatctggtcagatacagtaatatacattcattcctttggtctttcgaggttttatttat

aatcgtctgacttttgcccctataaccagtgtgaatttaacaggacagcgataaagtcaggtgcatcatgccacaaaggaatgaggtttgtaaagaccttttgcaaaaaaaaaagaaaagaa

aaaagaaaagttgttgaagcttatcagggtagacaaggttacaagcaattcctaagcatggctccaatgaactggccgtcctgccagaattattccaagattaaggtataatattatctaggaa

atgacaaagaactgcagagcaacattcagagatctctctcaccttggctaatgtcaaaataggctctatgccagagtagcaaggcagaaaccaatgttgaccaagaaaaacatagaagcgt

gtctcacatttgctaagaagcaccttaatgactctcaagaactctgacacaatgccgtatggacaaattaatcaaaagtaaaacttgttatgcatgggtgtgttccaccattatgtttggtggaaa

ccaaacagtgcatttcatgagaagagcctcaaagcaatagtcatggtcagtaatggtggtgtggtgtttagtgtgatggttcggggaagttttgctgcatcaggacttggacaatttggcatca

ttgaataaaaattcaaccaagtctacctcagaattgtgacaagattcggaatggcctagtcaaatttcagacagtgatacggaagctaaatgtggagtaccataagtagtaccagtaatttgcc

atataaactttatatatatatatatatatatatacatatggtaattaaatattataaacaataaaatatataggcatgacattccaggcattatgtggtggcaaaatttcatgtctatataattttagtttttt

actttaaacttatcagaattcacacagcattcatataattttgcttagcattttgtataaaccatttttcaatgtatgtccctctccctggatctgtcatcacatccctgtcctcttttcctgttaatgtgtg

ggagaagagtatctgtgaagatgatgaaaaacgccaacacaatgaaagtcactcaagactttccactttgtctctcttaaatgtctcttgctcaaacaaccttttgcactttcacattttgtttagta

tgtggccacacagttattgttgtgggtccttactgggtctgtcctctgaaaaaccacatcagttatatcagactatatgggctctgtggttttggttgtgtttcttactgtcgatgccaagttcttgat

ataagtataatctatcattttgtgagcctcattttttccattatcctttcaaaaaggcaacagcatttgcagaggacacatttagaactcctgaacagttgggacctttgcaaatttccgctttcataat

tgtaatacaagcaatgaaaactgccaaagaatgccattggcacagtatatattgggccacgctttcatgggcagatttcagccaccctaatgtattttagcaaaagtaacaaggtaatttacag

accagaggcattcaatactgatgacctttaatatatctgtcatctcggatgtttttgctgctgagagacttctgttgttggatccacctgtgaaagttgttaatgaagaagtctcagcagcatgtga

aatggccactttcaaatctgatgtgctgatgttaaaaacatctcaaatgggacctctggaaattgctttcagtatgaattactcaggcatattgtgagtgagaaaacgcctggtgttaagccatca

ccatgtgaaccctgaagctggaactgcactaaagcaaattctctctctactcttttctctcttctttttttctttttcttttttttttttggtaggaagccttcactagaggtcaaaagtcatactagatgct

accagtactctatttcatactttgcaggtgggttttccaaggttttccagtcaattagagcagagtatgtagcacaaggacagggtgtacctcagggatctgtcttaggccctttgaggttttctc

aagaggctgagttttaattcgtaacgcatgtgtaaggacagcagaagagtgtaactccctgttggaagaccacagatcagagaagttgctcccatgtgatgttggaaaactttaaacagcttc

tgtaattcaccaaacagagaaaaaggggtagtctttacattaccccgaaacccttggtgatccctaattcattcctctgctccacgatttagttaactccatgactttctcttccttagatggaagt

gtgtgttcctcacccagggaagcatggaaatacaatcagagcaggacacctcaccctgggcccacaagtggggcatccggctggacttgtacttacggcaagaagtacagcccatgggt

cgtctataggagggacaagagaaagggttgtctgtgactttaagggaagagtagcacagcagaatgacaaagctgtcatctgacgagaaaacacactcttgacacagtttcctctttttcct

gcatgagtggctgttagcacttaaaactgtggataaatgaatttctgtgatgtcctttgctgtgggaacagcttgaataaataaagaattagtattctaccagaaatagtgaatcattattacagca

catgccctgcacatctggacgctctgtgaagtggtgctgtaatataaatgcgtatgtatagaccatatgggtgaattaggtcagtcaattataggctattattgagcatacatagcttaccacatc

tttataatgtttaggtatgtgaaaagaaaaagaaaaagttaatgttactggttacatgttggtctttcactaatattgtgtcatagctaaagcaatagaactaaatgtttgattataacagtgcatggc

tattatattgaatagcacattattatatcatgtcatagtattgtaaagaaacaatgtatttttgttgtctctgaaacagtaacacttggcttgattgtttgttccaaatccaagtttgtttacacacccatg

cccctggaggtctcttttgacattgtacctttgcatctgaactgcaatgtttgtaccatcaacaagagtactagcttacagcagtagctgttttccactgtagctaggcaacgactcgagaggag

aagagagttttattgaaatatttgttcttctggatttaccacccaatctttatacacagagaacaagacttgagtttgtctgccatggatgcactgcaagagatgcgaataatgaaagttgctttcc

aaaggcaattaatttagagacgattataagcaaataaaatctgacatgactaataacacaaacaaatttttcacttttcacatttttattgactacattacactgtaagtagttattcacattgcaagg

tggagaaagtaagtgttaaccctccaccaaacattttctgtagctgttcataagactttagataaattttggaccatccctctacacaaatttgtttcagtgtatcagtgtctctgggttgcctgtttat

ggttatgccacagcatctcaactgcttttaaatttcatttttcctgccattccttagttggtaatgggtccttgtcttgttgcatcaccaaacctctggacctcttgctcttgaagtcatgattttgctgtc

ctgacattctcctgtaaaaggtcttgatatatgtatatatatatacactaccagtcaaaagtttggaatcggtaagatttttaatgtttttaaaagaaatttcgtctgctcaccaaggctgcatttattta

attaaaaatacagtaaaaacagtaatattgtgaaatattattacaatttaaaataactgttttctgtttgaatatattttataaagtaatttattcctgtgatcaaagctgaattttcagcatcattactcca

gtcttcagtgtcacatgattcttcagaaatcattctaatatgctgatctgctgctcaagaaacctttattgtgtacaattgtacaaaatatttgtgtacaatattttttttcaggattctttgatgaataga

aagttcaaaagaacagcatttatttgaaatataatcttttgtaacattataaatgcctttactgtcacttttgatcgatttaatgcatccttgctgaataaaagtattaatttctttaatttctttcaaaaaaa

taaaaataaaaattcttactgaccctaaacttttaaacggtagtgtataatgttacaaaagctttgtatttcagataaatgttgttcttttgaactttctattcatcaaggaatcctggaaaaaaaagta

cacaactgttttcaacattgataataatcataaatgtttcttgagcagcagatcagcatattagaatgatttctgaaggatcatgtggcactgaagactggagtaatgatgctgaaaattcagcttt

gccatcacaggaataaattactttataaaatatattcaaacagaaaacagttattttaaattgtaataatatttcacaatattactgtttttactgtattttcaattaataaaatgcagcctcagtgagca

gacgaaacttcttttaaaaacattaaaaatcttaccgtgtccaaacttttgactggtagtgtatatatgcattgttccttcaatggctggtctatacccggaggcagcaaagcagagccaaaccat

gatgctccttccaccatgtttcacagttaagaagaggtcttgttgttttatgcaatgctgtttttcctctgttgtgcatttttgccaaaaagttaaacttttgtctcaactgtccaaataatattttcccag

aaatgttgtggaacatccaggtggttttgggtgaacatgagactcaatattttttttagacagcagtggtttgcttcatgttgttcgacacatgaacagacttctgtagtttgaagagttttctggat

gtttttgctgttatctttaggttctttctcacctcttttaggattgcccgttgtgttctaaaagtgatcttgacaggatgtccgcccctagggagagtagcaacagtcctgaatttgcaacatttgtag

aaaatttactatggactgataaatacacgtctttagtaatgcttttgtagccttttccagatttgtgcatctctgtaatgtgtttttaaagtcttgtgaaagcttttgcgatcgaggatttggtatacactc

accaatttttcttgagaagaacagatttgtcagtgtgtgcctttattgaaaggtgcaggtcatctgtggacacttccacacttccaatctcatttccttaattgacaccaattagcttttggaggtaac

acagaggttcagcctgccttggcaatgatcatccaatgtcttgaatctaaatttaaagatcaggccacattttatgggtcatcagagcaaaaattcagatatcacaaacttattctttccactatat

agtaaatttgtaacccagagtgtgcaaacatgcatgttatatgtcaatagcagtttgtcaagcacattaaagcatttcattgtatttctgtgtgtcttaacagtggaaaacgacagggagctgtctc

cacagagacgacaccataaagagttcaaattcaacctatcccagattccagagggagaggccatcacagctgcagaattcaggatctacaaggagtgtgtcaccagggccttccgcaat

gagacgttcctacttagggtgttccaggtggttggggaacacccagacaggtaaagaagaagatcaatttcaaaatgctattctataaaatcccatattctttatttgctatactgtaaagtctga

ccagttgagtacagttaaagaacttgtataaaatcattatctaatagtaattatgtaacaatctctttcaaaagtgtgtaaagcacaactagaaaattaagttcacttagttgggtcttctaacgcca

ttaaaacttacatcttattaaaatggtattggtgcaatgacttcacttttaaataaatagtgcaccatttttgtgcacttgtccatggttttcaagctgagtaattgactgtattttaaatcaagcccagg

cgtccctcactgaacacagcagaggttgtctaccatgtactttatgtagactacggccacgaccacaacgcacaactgaaatagctttagtttggcatctaaaaaagaacccaccaacacac

gtcaaacattgactttgtgggtggtgggagggatatctccttgagaaaggcagatggcgtgaccggtaaagtgttcaaaacacaccaataaggtgtgattatgagactacccattttttttttaa

ccaagaaaggcaccaaacaagaaacaaaggcctgagccaaattctctggctaactgcatgtttcttttccaaatagctttctaatgtttatcacgaagtgttcatttggtgtacatcctgagttga

acaatggactcactgttttccacaaaccttttatttttagccatatcatgacattatgtatcgccagtcattgtatcactgtacaacaaggtttaccttgatgcaaattatagcgtgctttgtggtatttc

cctacatgccactttctgtcagtaaatcccaaatactgtgtggcttgcaaaatgtatttgcttacaaacagattaatttgcaaacttgtcaagttctaacagcataattactgtgtttatggtttgtgat

ctttaaaaaacacattggttggggaaatgcggtatagctaagctaagcagatgcacaagatggctgagagaaacttctgaagtgtgtaagcctttaatccatgaagcaaaaaataacttggtt

atggaggtggtgcagaggggctgtatatctggctgaatttagcctatgtggcaagacgatttgacctaaaacagaagtatagtgaacctcgagtcaacaagcttcacaaggtcttaaatgtaa

agaaacaatggcttgtgagagcatttttcaagtgattcccaacaatgcacacattagttgtttatctggaccatatttaatatgttaaaggtgttccatttttgcaccactagacataaaatctattttc

agacactcatccatattccattaattgcttgcatactgctgtagcctgtctattattttaatggtaggaatcagtatgaaaaataaaaataaaatgaaaataaaaaatcggcatgccaaattgttgtc

acttgacttcattacatggcatgtttaaggtagtaattggtctagattggaaacaaaaacaattatttcattgtttgtgtcaaaaagtgtcttctagcagtctggtaataaataaagggtcactctca

ggctttacttactgcagtgttggaaactgtattcttcataatgttctctgttgtataatgcacactttggcaaatatagtaggtcatcataagtattctttgcataggctacataaatcaccatgatatg

ctattttaagctattgcaatttgaccctttgctgagcctggcttaaaaaaatgtcagttactgaccaatcctatttttctaataaagtttatggagtatagttgtaaacacaatgtagattaagaggct

gtttacacaacactattttcaactaaaaactcaaaacgttttatgtgttttggccgttcatttacacgacaaaggagttttgggggcctgaaaacgcaaacttttgaaaacagctttcaaggtgca

cgttttttaaaacgataccattattgtcttcgtgtaaactacaaaaacacgaattcgtgaaaacggtgacgtcatgcacatgtgtattatgtgtccagtctagaggtgcgtagtgtttctttacaaag

tgacatcgccaactactggcctggcatgcataatacagcgtttttagtcgtttttgtggatccgtgtgaactgggatctttttgacaacattgtaacctgtacatacggagccccgcacatgacat

gcaggaaaaaaatagtaggctaaatcgtgcacacgatttactaatttgtctccttgatttataaatcgtgcacacgatttacttatttgttccctcgatttgctaaattgtgcacatgattttcttttttttt

cttgcatgttatgtgcggggctttgtatgtacgtggaaaatgcaaaggaaaaccttttctgtttttagtactacattgacatgcaccctcaactgtttgttgcccctcatttaaaaataaacatgtag

agcaaaaaaattacaattgaatgtaactaaatgctatttaatttaaaacattgattcaggtcatgttgactgtaactaaagagttcacagtaccatagtgaatgtattttagaaaagtttagttcactt

aaactacttttttaaacacttgtaattacattaaaatgttttttttttctcttttaagttgaccttgataagcgtttgccttcattttgattcagtttgatacacagtaatcacattattgcataatttaacagttt

ctgtatagagaagtaccagacaaaacctttaataatgggaaactagataattgacatataatgaatcaacatactgtacatttgttgtattacaaaaaacttggaatcaaaagaaaagttgtattt

gagcttatacgtttacatttacgcatttggaaggcatttttatccaaagccacttatagtgaatatacattttatcagtgaatgcattcatatgttattatcttgggaaaaaaaaaactgttttggagaa

atctgagatgcatttttaaacactgccaattgtatttattttaaaatcaggttctgttttcatgacgatccatcaatacctcatgaaaaatccagctagccatgactgtagagttattacttttctgtttgt

atatattctaatccctggttgttttcatgtacatctggcctccttggaaaaccctcccaagcaatatgtgaagacggtcctgtatgcatgcaatggcagcgtgaagctattaaactataggcctatt

ttaacagcagccaatgacagaggcaataaatgcgatttatgaaaccaaaaaaagcccctgacacttctgactttattatgccagttaatttaatgtgtctataagaggctttccactggagctttt

agagtcggcagctcccctgtggtcttattcggaccccgtcccaggtgaagctgtgtgactggtggcactaaacagtatgagtcagcataagtatgatgcgttcatgtcgaatgtgacgtgtg

aaccaccggagggaagaacctcatatggagctccttagagtgcagctctaattaatgagcaataacaaggggctgcagggacattcccagaacacctgtgtgaccgacagctttgaagtg

acccggccaatacttattaccatgcatagtgtgtaattactataatgggacaggcctccctgtaaggcttctacctctgttttcaactcaatgacttgaagctcctctgagtttgacaaactgttatt

tggtttaatattgttcactgttgaagacatgccttaaaggattagttcacttcaggattatttatttatttttttgatcatttactccccctcttgtcttcccagatgtttttgtctttctttcttcagtcgaaaa

gaaattaaggtttttgaggaaaacattccaggatttttgtagtggacttcactggcgtacaacgggttgaaggtccaaagtgcagtttcagtgcagctttaaaggggtctacacaatcccagat

gaggaatatgggtcttgtctagcgaaacgaaaattaacgtcattttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaatatatatatatatatatatatatatatatatatatatatatatatatatatat

atatatacaaaaaaaaaaaaaaaggtaaacaacgatgtcggacgattttggaggagaaaatgagatggagtattttgccctacctcggtacttccgcctacgtcacgcgtgacctttccagc

gagattacttaatgtgtgccgtgatgcatagcagagctagtgcaaggcgagtatttgtggtcaaaaaagtacaaattttgacagatcttataaaaattacaggttgtttcgctagagaagaccat

tattcctttatatatacatatatatgtgtgtgtgtgtgtgtgttttatattttttattattacaaataataaagtaaaaatatatggaataaatggtaataataataataataatgcatgcaaatggtagaaa

gaataagtataatttaaataaaattagaccagcctaattatactgtatataaagagaatattaatacatttatgaatatcaaatcaaaaatcaaattaagctggtcttattttatttaaattggaaatatt

atttttaatttaatattatttttaaattaaattattcataaatgtattattacgattaattagaacaattctctttaaaaccaatgtgtttgcaaaatacagttgtaatttcttctaaactgcatctgcatttgctt

tgacagctaccatttatacttattctttccaccatttgcatgcatattatcattattattattattactatttattccatatatttttactttattatttgtaataataaagaatataaaacactagtaaacagtgt

cattgattcatgtaaatagtgtttataacatttattgatctccagacttgttcatttgcagggatgccgacctatttctgctggaatctcgaaggttgtgggcggctgaagaaggttggctggagttt

gacatcacagcaactagtaatctctgggtgatgagtccacatcacaacctgggtctacagatccgtgtggaaaccagcagtggtaagataagtttaacttaatactggacgcctatgagtca

ctatgagcaaagatgtttcaaatagattatttccagggagtcctgcatcaaccaaacaagtgccagtgagatgatcgggctgatttttgatttttggctgggtataatatagtcctcattgctcagt

taaactttaatttttaactttaatttccttttttaacacagttcagttctgtttgatttgcaatttgcaaaaaaagtcatataaaagaaacatttgcatgtaaccacaaactttggtactatgaggactgat

gtgaccatgaacaaacagcacttatcaaagagattccctacttattggacaaggataccccatctattgtactttcttctctttgaaggatggagcatcagtcccaaggaagcgggcttagtgg

gtcgtgatggcgctctggagaggcagcctttcatggtggcctttttcaaagtaagcgaagtccgtattcgcactgcccggtcgacaggcagacgtcgtcaacagagccgcaatcgctcca

acagtccacaggaggcgacaaaaggatcagctcatacaggttagagatttgtatttcccatattgtgaaataagccccttcctattttcttttctttttctaaacaatacaagtttaatatgcacgtat

ggcagaaatcagccatccagtctcttttaatttttcaggagatgttgacctggatagtttggggatgttttcgagtctgatgagttttattgagtccagatataccccttcataatgaaaatctggac

tcaataaatatctttaagattttttcaagaattcatgtaggaagcagcacatatgtaggcctgacatcatgtgtcctgatttgaagtcttttgccaggcctcaatctgaacctctaagagataaatag

aaaaaagccctttttaatattggtgtgaacatgggacacctaagcatttaggaacaaattacaccgtacgtgaatgcaaaaacacttctaaacaagcttgatttcatgcattgttgcattacaaaa

tgcattaaaaaacacaatgcaagtacacaatgcattatcagtgttgccacaaggggatctataaaaattagtgtaaactgtctttctttatggtcagtttcctttttcaggccaactagcttaagttt

gctaaactgttgctgacatgtttatagctgattaatgacacatttggttgaatgacacaacccagttggccccttgagtgccgaggattggtaaccactagaatgcaaagaacgaactggtgtg

atgtcatttcaaacaaaagcagaccaaaacaaagtttgttttgttcgttttgggagttcgaaacagcttgccaaagcaaacaaaagttggtaaactatcattggtccttggtgatcacgcaatat

gtaggagtggctctgagactccgccttctttgagatggaaatcttctccgtgtactacaaatctttaaagtaaagtttatttttaaagaaaaatgagatggagtttaaagaaaaatctatttgcagat

aatataatattaatgaaataaaaggccatttaagtgtactcaacagaagtacactatcatgaatatgtttcttttaaagcacccctattgtgctattttaaaggttcttaattttgttttggaggtctcct

ataacaggtttacatgcatcaaaggtcaaaaaaaggcttgcattttctcataatatacattgcacatcaccacattttttaataaagctcaaacgactcgtccaatgattcagtctctctaaatccct

cctttccgcgagcctactctgctctgattggccagacggcccagtctgttgtgattggtctaacgcttacagcgcctgtcggaaacaaatcgtccattagcatatcagattcagcaccatagac

ttcctaaagctcacactgtaaagacagtaacaatggcgtcaaatttagcttatcaatccgggctcgagtccagtgacgaaacattggaagaactagttattcaacctgagcctccatcgcaag

aacgacttgaccaggatgtttctcagttatacgctactcggtttaacataaaatatgagatgttgcaacctcaccatcagcattaacaagtgtatgtccatcaacaaaccgatatatttctaatttat

tccggtgcacatgtgggaactgcatcattaactgtatcaataacagcgcatacgttagccaaacactaacgtgaatgactgatgtaacattaaagtttgtaaagcaaatcttgctccatccttttt

cattactcaaagtagtgagaatgacagacaatctgcattaatggacacagttttaggtaatagtgggccccagctgatttccagaagtatttcagcaagagagtaataacgtgggttagccag

agacctacgcaataactacgtattttgaacacctggtagaaaaaatatacattgacaattaatcatacttacaggttgtgattctgaggagcaagttggtccaaataaagtgagtactgtcccat

ctttaatggataagcgttttgtaaattcctctcgtagatttgaaaagcagtcgtcaaaaggttatactgctgttgtattgctgtgttaattttaaccactgactcttcccatcctcatcctttggaagtgt

ttacatagagaatttgcttttgcagtgctgaaaacagcatcttctcaacatacacaacacgaaccaactacagtgtttgagggcgggtcaaagaagaagattttcacatgcagggcaatgaag

accatgggctggcattaagcaaatgtgttacagtgttgcgtatgcaggttacggaaacgagactggaattcctgacgactcttttgggagacaataactttattaatcgagcactttcaacttta

caactttgcagaccgtttacattcacagacattttctttgacgcgttttccatcattatttgacattgtttttccctcaaaccctgtgactgtacaaagcatgtgctattcagtttctgattcagggtgtc

tgttgcttagcatgtagacctttactgaatgtgtgaacgctgttgttgtgtgcatcacatccattagctatgtttccatccaaagttgcaaatttaacttatgcgcaaaattggaatatcacataaaat

atttgtgatttaaaatgcaatttccaccccatgtattcaagagaacaacttcctaggaaatatctgtagtaaaaatggaagttgctgcaatcggggatgccactgtggctctttttttttttttcatatt

ttataaataacagaaagtgcagacaaaaagcaataaatatttatcttagtctcatcactccagagtatagagtcccagtagtcttcatctttatcagcatgggccctggcaaactctaagcgggc

ttttttgtgcctaggctttaggaaaggcttctttcgtggacggcacccatacatgctattcctctgcagtgtacgccgtgttgtgtcacgggaaatagtcaccccagtttggctttctacttctttag

ataactgcagtaaacttgcatgccgattttcttcagcccttctcatcagaagatgctcctgtcgaggtgttaacttccgtggatgacctggacgtctctgtgagatggttgcaattccatctttttta

aatttttgtaccacttttgctacattattctgactgataagtaaagctttgctgatcttcttgtagccttcacctttctggtgtaaagaaattattttctttctcaggtcttgtgacatttctcttccatgtggt

gccattgctgacagcatgaaatgggaaggggttttaacactcttttatagtcaactgtctgctggacacctgtgtaatgaataattatgctcacctgtggttaaattcttgttaaattagacatctgt

agtctaaaatttagctttgctccagagattttcagtggggtgtactcattttagcatatcagattcagcaccataggcttcctaaagttcagcgattgtacacagtgtaaagacagtaacaatggc

gtcaattttagcttatcaatccgggctcgagtccaaagacgaaacattggaagaactaggttttcaacctgagcctccatcgcaagaacgacttgagcaggatgtttctcagttatacgctact

cggtttaacataaaatatgagatgttgcaacctcaccatcagcattaacaagtgtatgtccatcaacaaaccgatgtgtatatttctaatttattccgatccacatgtgggaactgcatcattaact

gtatcaataacagtacatacgttagccaagcactaacttcatctttgtcagcatgggacctggcaaactctaagcgggcttttttgtgcctgggctttaggaaaggcttctttcgtggacggcac

ccatgcatgccattcctctgcagcgtacgccgtgttgtttcacgggaaataatcaccccagtttggctttctacttttttagataactgcagtaaacttgcatgccaattttctttaacccttctcatc

agaagacgctcctgtcgaggtgttaacttctgtggacgacctggacgtctctgtgagatggttgcagttccatcttttttacatttttgtaccacttttgctacagtattctgactgataagtaaagtt

ttgctgatcttcttgtagccttcacctttctggtgtaaaaaaaattattttctttctcaggcttgtgacatttcttttccatgtggtgccattgctgacagcatgaaatgggaaggggttttaacaccctt

ttatagtcagctgtctgctggacacctgtgtaatgaataattagactcacctgtggttgaattcttgtttaattagacatttgtagtctaaaatttagttttgctccagagattttcagtggggtgtactc

atttttgcatcactctaatttgagtaaaactgttatattattaaccttactttcatgttataagttaaacagatgttatataaaacttggtcttgtcaacattttggaaattgtttttgtgttcattgagatatt

gtttaaaatgttacttttcaaagggggtgtactcatttactctgagcactgtaagtaaaaatgcatacacctttctcgctattgcttacatgccagtgattacccttccacatgccataggacgccta

cccctgatctagctgtaacattctaacactgcatcgtttcacacttcacactacagtgcgtaatgaaccctgcaaaagtggtgctaaactgttcctctacccagggttaaaagtactgtttagaac

agcgataaccctgggttaagccacggttgccaactctcacgcatttggcgtgagacacgcttttgggctctgtctcacgctctcacggcacccaaaactaaatctcatgccaaatgacaaga

caacgcaaaataaatctgctcgtaggagttttccaccagcacatgacttgatttgtccaaaacattttttcttgaatcgattgaactgatatgaagtggcaagattctgccccaatcaaatttgttc

atgcacttcgcaatgagatcaaagcagtaccagatggagaaaatgaagaaacggcggtgagacgggtatctgtgcccacaaatcagggtactaaattgacacatgccaagtgccaaatg

cgattttttaggagaccataaccccttatttattgtgcactttgatctttaaaactttgcagacattttacattcacaaacagctatattacacactacatgaaagataatattcaaaaaagcataata

gcaacgctttaaatgggtacctttctagtttaaataagaacagtagaggacagcaggatcttaatccagtttctgcttctcattattaccctggtttggggtggtctgaaagcttaatgctaatacc

tcacataatccagagctaattgtgaatgtgaggccttgaaacataaaggaccttaaggctgaaaatggtatatttatcaccatttatcaaaatgtatgagttcgtaagtttctttcacattcataatt

ggatgaatctgaattggaactactgtatgttttcttgatgtgttttaagatgcatatatttttatttcttttgtcaggaacagtcatttctctctgctttaatttaagaattatttgtgtttgtgaaaggcaaa

acattcagtgtatgtttcgactgcaatacagctaattgacgtaaacacgcattatgcatgcaatacatcctgctgagaataattttcatcagtgccatatgtgagtcagacaccacaatgtttcac

ccatttatacagtagtgcttaaactcaaagctcctaaacctcgcaaactacgtgtctcgtctaatgaggaaaatctgcttaatttgttctgaaggactggactttatgtaacccaaaacatcagcc

cttctcctcggctcgggatgtctggagtgtaaatatttatatttggagcaggaagttgcgaggccgttgtgacagccgtcccctcgtggcagtgttgagaaagcatatggttccaatatgaaat

cactcttatcaaagcattcatagactgcctgtgagcattgcatcttgacccatattgagagatacaagtgcactagtttataggctctgtaattgctaattccattttagagtcaagagaaaagaa

aataacaaacactatttaagataataacatttgtttgcattttgacataattatcatgacaatttatgatgataattatatattatttaaatgactaaacattaaataaacattatataatatagttgtatttg

gaaaaaaaatttaaatgctgtcataatgaaagtgacatgtaaaggccaatatggtaaggtatggtcctctgcatttaacccatccaagttagtgcacacacattaggagtagtgagtagtgaac

acacacacactgcaaactatagacagtaggcagccattttgctgtggtgcctgggaagcaattgggggatagctgctttgctcaagggcacctcagtcatttcctgctgtattgagaatcgaa

cccacaaccttcgggttaccagtctgactatctaaccattaggccatgactacccaaatgcatgttgtgtaatgcatgttgtgcactgatgcagtcattcgattatgtaataaatgaatcatccct

gttgatattagagatgaatcaaaaattgttttcattaattaaaaaataaaactgaaataaaaaaaaaataaaagtacaaactataagtaatgttgtaacatgtcaacttaatctactaacccaaacc

ttaaccataacagtctactgatattcgaatgagagttaattgacatttagttgcaaagttacttagtagaatgtaaagtgttaaaatatataattattagatggaaaaaaaaattaattaaaacaaaa

attacattgccttggcaactaactaaaaataagctgaagcactgtaaaccctaacactcaaaataatgaattggtttaagtaggacaactagttaaacaaattagtacagtcaaatgaacttttat

gagtatttagcacatactttttcttgtttcattgttttgagtttcatgaactcatttcttttaatttagacatagtagaacttaagtttaattgaaactgggctgggattattagtcccagaatgctttgccc

atggcactcaaaagggagagtaaatgttaaaattaagtgttatacctgtatgatgtttttatgcaaaattaatattaagtgggagatttactagtgattagagttttattatgctaatttagaagggttt

ctgtgggtttttttggagagttaccatcatggtgacaagtggttcatgcggcttggtttgagaacaggtatattaaaagttttatagtgctgctcttgcaagtctaccatgctattgctaacatgcta

gtaaattagcaagttaacattttctgaattagcttgttatagctagctaagtttccactaataaaaatgtttctgttcagattacgtgttaattttaagttgaatgtatgaattattttactaaaatatttcag

tttaataataattaaaatgtatgaggacaaaataaaaatctgcaaattctgcttacttaaactttgaatttcttacttaaaaactgttatgtaaccgattgctttttttttttttgttttgccaacttattcggg

tttacagcgagtactcaaatgaataaaactgtaataaaaataaattaaagctaatagaaatataaaaacaaattgtacaaaagcacataataaaatgacaaatctttaaaattaagattaaaatg

gaaaatagaaataaaagctaatttaagaccctaataaattctataatagtaaataaataatactaaaatagcactgatatgattgataaaaatgtattaattaaaaagatacccttttcttttttacag

cagtactgcaaaatttttataataatttttttcgcattacattattttttttcgcattacagtaatgacaatgatatttttgcagggaaaatgtgcttaattgtcatgaaaataatgtcacaatgcaagaaa

tcttaatgatcctaaaaccagacttcatttatcttttctttcttttgattcttgggcagaaaaatgacatgtttcaccctttgaaaattctttctgtctctatgagcttccaattcagacgaagcacattac

gtgcgattagaggtttgacactgtttctaaagctttggctatctgtgaaaacaaaacagcacattaagcgctaatgctcagctcgaagtgcgggggagctcgccggcctttggcctgaggga

agttaacaagcgtctagctcagctcagtatgtgccgccccagcgctagacctgtaattacctccgtgctgttattaggtgctgcgctaaaatgcctgaccacgttaacagtatcgcttaaagaa

ttataataacatggcactgtccagctgcgggttttcatctccccctccctcagccctccttttgtttaccattgaggtaattagacatcggcgagctctaaggatggaggtttaataaccacagtg

atgaaagacaggaagagagataaacttcacatggacgcagacagcactcacattctctttcaagtcatcaagggagtggggtgagacagcctccctgcctcagctctttccaggatggatg

ttattccatgcggaacgccgtgtccattactccagcatgccgtcaccaccaggaatgtcattgtgtaattgctccaaagagaagctttttgtgagaggcatctgtgagatttcattgcagttacc

atgtttttgtcttttggtcaagaaaacgcatgtgtatcttggcatgtgattatgacagtttttggctgtaactgtcaaaccaaaaaggtcataaaactaatataaccatttctattaaaagttcttgaac

atctgcgtttttccaccaatactttgacagtgaatcctgtgcggtgacagagctgcactcatgactgcacatcatgaatcgttttaccatctcaattcaagccgagtcctcatcctttgcaggattt

gctttcgtagtgacatgtcgacaacaagaaccaaactcttccaggcctcagcaacaactacagtgtttgagggcagggtcaaagtagatgttgtttgttgaaaaggcattaggcaaatttgtta

cataacataatgcaggtttatgcaggaagtaagactggaattctagtttcatttcagaaccagttcttttgggagataactccatttataccgcagtttgatctttgaaccttttacaaattcacaaac

agctatattacatgaaagttaacatccgaaaaagcatagtgagggcactttaaagaattgtttagtagaaaatcaaatcagaaatagaattattaaattccttatgattgtcatccctacatatgat

aaggaggcagcggtcttcactcgagggccagcggaggcccacaggacccactcacagcagtaacagtgtggcattaaattttacaggcaggaagagaagcgctttatggggtaccaca

aatctggcctttacactcaatgaaactccacactaaatatctgctttactcctaaagagcccttgactgtaaatattggcagtaaattagcaaaaaaggaaaggcggagctaaacaaaactggt

gtgtgtgaatacagtctgaattcagtgtgttagtggtgcatgttttgcacaataactgttccaggttaagaaattatactctgcatgaagatgaagcctgtttcagaaacattatgatgttttcttgac

atttcatgataaattctcatgactgtaatatgaaaaactctcttgttatagcattatacagccagggttttacttttgtgtttttaattctcataattctcaataatgaatacagcccagacaagatagac

aagctattaatattattggtaataagtactggtcctaaaaatggctttgttatttatgctaattaaaatacctttagatttttggggagaaatatgacccagaactaagtttcatgagattaaactattta

aaaaagtggatcaaaagaagaaaaagaccaatattcctcttaagaaatagcaaaatgctgtttaaaatagatggagtatagcatgtcacagcaccggacatgtcagcttcccatatgtatgag

ttatgtgtaactgtgtcaaaatctaccattcttgggttgtagtttggcctcattctcaggcctgtggttaggtgtagaaaattcttgactataaatgtcacagttgggggtctgtgtaagacaggttc

actgctcgggatcagagaggaaagggacccctgggggaccgactaaaaggccactcgggagagctcgggggccactcctgctggcggatttatctcagtaaatatttaccctagtcctg

ccagacagagtgagagaaatacaagaaggggtcttactttgggggctaaagaaggccagagatttcacactgtatctcaagctcatatagtttaaacaaaacatatttgaattaataatcttta

gtcaatactgaatatatttatattctctttcaaatgtgaaaattaccacaaactacaacaataatttgttattgttattggggctaaaggcaccggggtaaaaagtgcatctgattttttgttctcctcta

aaccactagacacttattgcagcactttcctaaatgtccgcgtttcactattcacattgaaatttggctgattcttgactcaattggtggcagcaaagtttattttgcactaatttgattcgagttctttt

aaaatgttatagatttatgtaacacatgacaaaatttaccaatattttgagacaaaagtcttgataatgattttcagttttaatcaaggtaattaaatcaacagttttcccaataactgaaggatatgta

aaaggtatttggggtaaaaagtgccgctgctattggggctaaaggcacaataattgacatcaaaataaatagcttgctgacattttttacatttattggcatgacatagttatattcagatggttaa

tataatttattcagttggttgaacatatttaatgacagtatatttattaaatacaatcacagctttaagaccaagaagcttttgccaactagctgcatcagcaggattttcaacaaactttctcttaaag

gagaaagtgggaaacaccttagccctggatgattgttgagtgattgatttaggatgacctcagagatttgtctacacactcaccatgtggtttcagttacagccttgatttctactctacattactc

atagacaatcatgtttttaatgaaggtaggttttttttagatgaatacaggtttctcccattttattagaatcatttgtcccttttgtaaatgtgtttgtaaagtgaactgtccaaaaacatttttaatttgaa

agatttttgctattatctcaaaaaaagttcctgtctatgaagccctattccttccaaaaagcgcaatgtgctctgattagtcagctgaaccagtgtgttgtgattggtcaaccactttgtgtgagtttt

ggaaatgtcacgccccttaccataactgcaagtttcaacaaattattaccgcaactcaaccatatgccttggcaggaattatttaaatgaggaatattgtgatctgtatgttcccagaagaaaact

caagactacaatcgaggcatttcagggagtccagaaacagtgcttattgatatagagaataattccctttgtagtgtactttgtgcctactaactttaaagacccaggacctctttaagctcgtca

ttaaaccaacatatgctcattggaaaaacatgcctctacctacatttttgcaaaactccaaacatgtaccaaaacatacaagtttccagctgaaaagaacacttgaggcagtaaaacaccaac

aacttttattccttttcacgcaaaattatgattacggggcagattaacgattaataaagacattttttctgcagttccttgcacaatactatgttatgacctcaaaacccttttaccatagtccatcattt

gtaaactattacatgttgaagtttgcatcgcatgatcagctccatatgtgtggcttttctggagcaaacttgctcagtagctcacctggtacagcattgcacttaagtaaatgacatgatttcttcag

caaatgtgtttcattattaacactagtgggaatgtttagggtttagtgtaggtggaacgttattttcaaatgcgattaagagccgctcactggacttttaatttccgaactgtcgtgacacctgcaa

caatgaacttaataaaacatcaccagattcatgttcatctgtttcagataaaactaaacatgcttttagcaccactcactgtacagttcactttaaagtagaacaaaacttgcaagaggcatcgta

acatcatcttgcagaaatgttgccacaggcacacttttccaatgtgcctgagttggattaaacattaccaccaggtttctgactgacttggatgttgttagtgttgttgagcccagctggactgtg

atgttgtgcggaactgccaggctggctgagttgtaaatattgtgtatattcagaagaaaaggggcccaagcatcaaaccttggggtaccccagtcattagctagtgtgatttagacacctccc

cagggtggtttcaatctaatggtgtttcctgcaaatgaggaaccagtgtacaaacttaggggaacaaattttgtgtgcagtaagcaaaataaagatttgttgtgttaatcatatgtacattatcaa

aagcagtaaacatagatttcaacaggtttatgtattgtttttctcagctgtattatgagacaggttttcattgtcatgttgaatcaacatataatatttagtcttgcaagagcagtaaattaaatttactt

acatcataatgtcattacagtatggcttcattagggaggcttcaacagcctgtttgaaaagaatagccagcatgctttcagacagggcaaaagtgtgttgtatatgggattggggtgtaagtttg

atcaaatagatgaggtggcaactacccgtctcgcatggcatgtcttgtcaatgttttggttagtaggatctctggatggccaaagttctgctctttgggacgggaactcaaaaaaaacaggaa

gggattgcagctggcctcaactgatatcatagttttctctctcctctcctactccactgcacttagcaaaaattaccaaaaaaaaaaaaaacgtgtatgagaacaccagtggaaagaatgtgtc

agagtttgaattcagagcaggagtcagtgccaaagattaccttccccttcagaaaacccaaacctacctgctgctgacatgtggctaggtcacggggtggagccgctagataattgaaata

aactgtaaatgttgctgacagcaactggtaaaggacacattatgttgcaaagcaaatgtgttgtgacattaacaggctcacttcaaatattgtctttgttcttggtattgagacaaactttaaatga

aggaatttaatttggttgaaattggttaaaagtacaccaaagacctttcatgtgattggcaagactggtcttacttgccctcatgttccaaatctgtatgactttcttccgcacatggaccatgaaa

ggaaatgtaaggctaaaaataaaggctcagtcaccatttattttcactgtatggaataaagatgcaatgaaggtcaaaagtgactctcattctgcaatttggattccaaatttagtcatactcaact

cagaatgattcatttcaggtgtacttttgtgttagttagtttaaagcacaacaaaagattatcaaaacatcatgatttaaaatgtactttaaaaaaactttaatttgacattattacaaagtgtgcttttta

aaattgtaaatcattttcttaagaaacatgaaagtggactctctttaaagggttagttcacttcaaaattaaaatttcctgataatttactcatccccatgtcatccaagatgttttttgtctgtctttcttc

agtcgaaaataattttttttttttgaggaaaacattccaggatttttctcgatatagtggacttcaccgggtacaatgggttgaaggtccaaattgcagtttcaatgcagcttcaaagggctctgca

cgatcccagctgaggaataaggatcttgtctagcgaaacaattttattaaaaatatatcaatttatatacttttttaaccacaaatgctcttctcgcactagctctgcgatgtgccatgcattacgta

atcacgttggaaaggtcacacgtgatataggcggaagtaccacggtagggtgaaaaactccatctcattttctcctccaacttcaaaatcgtccgacagcattgttttaccttttttttgtaaaag

gcgcttgactccgggtacgtttacacgacaacgatatgcttaaaacggagacgtttttccgcgtacagaccacaccattgtggattttgtatgaatggagatggttttgacaatggtgtggtctg

tacgcggaaaaacgtctccgtttttagtccttttgactaaaaacgctgtattctgctggcaggccattagatggcgatgaaacactatagactgaacaagtaatacgcatgtgcatgacgttatc

gttttcacagattcacgtttttattgtttacacagagaccattttcaaaaacttgcactttgaaacccgttttcaaaagtttgcgttttcaggacaccaaaacgccgttgtcatgtaaatgatgtgtgg

cgcatcacagagctagtgcaagacgagcatttgtggttaaaaagtatatatatatgtatatatatatatatatatatatatatatatttttttttttttttttttttttttttttaatgactgatcgtttcgctacac

aagacccttattcctcggctgggatcttgtagcgcactttaaagctgcatttaaactgcaaattggacctttaacccattgcaccctggtgaaatccactatatggtgaaaaattctggaatgtttt

cctcaaaaaccttaatttcatttcgactgaagaaagaaagacataaacatctgggatgacatgggggtgggttaattatcaggaaattttaattcttaagttaactaatcctttaagtatactgaaa

atatattttttagaaatatacttttaagaacacactaaagtataaagtaaaatgtgtagtatgttatactgtagcctagcatgtgtaaatgtctctcttctctgtgattggcagattacaacagcagtga

tcagaagaccgcctgcagaaagcatgaactgtatgtaagcttcagagaactgggctggcaggtaaagatgaaggacatgcatttgcatataaaataactgatgattataaatgtgtgttgatt

ctagtttaagattagtgtaggtagcaattttaatttgaaatatgtcttttgtcaggactggattattgcacctgagggctacgcagcaaactactgtgatggagaatgctcttttcccctaaacgcc

catatgaatgctactaatcatgctatagtacagacactggtgagtccttcttcataaaataaataccagccctaacagaacaagtatttttaaaagtttacttcacagcagtaagtttataaaaata

aaaaaaaaaatcgtgctcccctttttaggtccatctgatgaatccagcgaacgttccaaagccatgttgtgctcctaccaaactacacgcgatctccgtgctttactatgacgacaactccaatg

tcattctgaaaaaatacaagaacatggtggtgcgctcatgtggctgccactgacacaggggacatgtaccctcctgcccaaccgctgcaccgcagacacacagacaaatgtgtgcaagg

actgacaaagtgaaataagacaagcgctatgaacaaatgtgagacagtgtctcaacagtggatccaggtttgctgcttctcctcatcttcacactcacggccatttctcatacgacatttcagat

ccttcaaactgccaagaggatgtacatctgaatctacagtatgaatagaaaatgaacccgatgtctacattctaaaacaaagacacaaagaaaatgtcaattatgagctaaaatgcagttacc

aaatcatacataatatgcaatatcttcatttttttgagttccacttaaaatgctacaaaagcttcaccgaaagagccaaattagctgttcttatactattttccatggtctcgttgccccttggaattaa

acggga

Design of the gRNA target site the appropriate target site for the gene of interest is selected on the website (https:// www.crisprscan.org); the GRNA TARGET site of the gene of interest is preferably designed in the exon or functional region; selecting target sites with higher scores, so as to ensure the target sites to be effective targets; ensuring that the target sequence is linked to PAM (NGG); the target of the target gene designed in addition can be detected through the website (http:// www.oligoevaluator.com/LoginServlet) to detect the GC content, the Tm value and the secondary structure of the target. The sequence of the GRNATARGET was synthesized from a 17bp T7promoter, a 20bp target sequence and a 15bp scaffold overlap sequence: (TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA).

Specifically, the target site gRNA-1 sequence, the target site gRNA-2 sequence and the target site gRNA-3 sequence in this example are all located on the first exon of the silver carp runx2b gene, wherein,

The target site gRNA-1 sequence is: 5'-GGAAGAGATTCTGTCCATAC-3', SEQ ID No.1

The target site gRNA-2 sequence is: 5'-GGCCCAACTTCATACATCGG-3', SEQ ID No.2

The target site gRNA-3 sequence is: 5'-GGCAGGTAGTAGTTCTGTAC-3', SEQ ID NO3

4. Primers for synthesizing gRNA

Some embodiments provide for the production of gRNA by designing and synthesizing primers and subjecting these primers to PCR amplification.

An upstream primer with specificity of T7 promoter comprising target site gRNA-1 sequence: TAATACGACTCACTATAGGAAGAGATTCTGTCCATACGTTTTAGAGCTAGAAATAGC, SEQ ID No.4

An upstream primer with specificity of T7 promoter comprising target site gRNA-2 sequence: TAATACGACTCACTATAGGCCCAACTTCATACATCGGGTTTTAGAGCTAGAAATAGC, SEQ ID No.5

An upstream primer with specificity of T7 promoter comprising target site gRNA-3 sequence: TAATACGACTCACTATAGGCAGGTAGTAGTTCTGTACGTTTTAGAGCTAGAAATAGC, SEQ ID No.6

GRNA-1, gRNA-2, and gRNA-3 share the same downstream primer Scaffold：5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAA AAC-3',SEQ ID NO.7

5. Synthesis of gRNA

The primers are subjected to PCR amplification, and a PCR system is as follows: 10. Mu.M gRNA upstream and downstream primers 2.5. Mu.L each, 2XPCR MASTER Mix 25. Mu.L, enzyme-free water was supplemented to 50. Mu.L, and a total of 100. Mu.L per gRNA amplification 2 tube. The PCR amplification procedure was denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, and 30 cycles; and (5) extending at 72 ℃ for 5min, and preserving at 4 ℃. The PCR products were detected by electrophoresis on a 1.5% agarose gel.

6. PCR product recovery and purification

After detection, the PCR products were purified and recovered using an Omega agarose gel recovery kit and the concentration was measured using Nanodrop 2000 (thermo scientific, USA). RNA was transcribed in vitro according to the instructions of the kit (TRANSCRIPTAID T, HIGH YIELD transcription kit (Thermo Scientific, U.S.) and then purified and recovered by phenol-chloroform extraction and absolute ethanol precipitation to give gRNA-1, gRNA-2 and gRNA-3, respectively, 1. Mu.L of the measured RNA concentration was removed and the quality of the RNA was checked by 1.5% agarose gel electrophoresis and stored in a refrigerator at-80℃until use.

7. Microinjection

Before injection, female and male silver carp parents are placed in a breeding parent pool according to the proportion of 2:1, and the female parent is subjected to induced spawning by injecting luteinizing hormone releasing hormone A2 (0.4 mug/kg), wherein the injection quantity of the male parent is half of that of the female parent. The effect time after the oxytocic is injected is generally 10 hours (the effect time is closely related to the water temperature, and the time is properly shortened or prolonged according to the different water temperatures). Microinjection was started 10min after the silver carp began to spawn (fertilized eggs developed to single cell stage after embryo water uptake started injection). The injection system was formulated using a mix of gRNA-1, gRNA-2, gRNA-3 and Cas9 protein (GMP-CA 9S18, acroBIOSYSTEMS), wherein the final concentration of gRNA was 2000ng/μl, the final concentration of Cas9 protein was 500ng/μl, and phenol red was added as an indicator at a final concentration of 0.2%. Experimental reagents were injected into single cell stage silver carp embryos using Picoliter Microinjector injector (Warner, PL-100A, USA). After injection, the silver carp embryos are cultivated by pond water still, and the water temperature is 25+/-2 ℃. During hatching, the embryos of silver carps need to be particularly carefully managed, water is kept to contain enough oxygen, water is changed in time, dead eggs are cleaned, and hypoxia or saprolegniasis is prevented.

8. Detecting target mutation rate

Randomly selecting 36 tails of the surviving juvenile fish, cutting tail fins of the juvenile fish, and rapidly extracting silver carp genome DNA by an alkaline lysis method. The fin is placed in 0.2ml of eight-row tube, 100 mu LNaOH is added for 20min at 94 ℃ and then the reaction is finished at 4 ℃ for 20min, 5 mu L of Tris-HCl is added after vortex mixing, mixing is carried out uniformly, and the mixture is preserved at 4 ℃ to be used as a DNA template. The amplification primers are as follows:

bmp6-F：5’-CACCCTCATCATCATCACCAC-3’，SEQ ID NO.8

bmp6-R：5’-CAGCATGAAGAGGGGTGCTG-3’，SEQ ID NO.9

The amplification PCR system comprises: 1.5. Mu.L each of the upstream and downstream primers, 2× PCR MASTER Mix 25. Mu.L, DNA template 2. Mu.L, and enzyme-free water was supplemented to 50. Mu.L. The PCR amplification procedure was 94℃for 3min of pre-denaturation; denaturation at 94℃for 30s, annealing at 58℃for 30s, extension at 72℃for 30s, and 32 cycles; and (5) extending at 72 ℃ for 5min, and preserving at 4 ℃. And sending the PCR product to a qingke organism for sequencing analysis. The sequencing result shown in fig. 2 shows that double peaks exist at the target position, and the efficiency of the bmp6 gene knockout of the F ₀ generation of the screened mutant silver carp cultured into the parent F ₀ generation is up to 86.11 percent. Where knockout efficiency = target position there is bimodal mantissa/total x 100%.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims (10)

1. The nucleotide sequence of the gRNA of the targeted silver carp (Hypophthalmichthys molitrix) bmp6 gene is shown as at least one of SEQ ID NO. 1-3.

2. The gRNA of claim 1, which targets a first exon of the bmp6 gene.

3. A knockout composition of the silver carp (Hypophthalmichthys molitrix) bmp6 gene comprising the gRNA and Cas9 protein of claim 1 or 2.

4. A knockout kit for the silver carp (Hypophthalmichthys molitrix) bmp6 gene comprising the gRNA and Cas9 protein of claim 1 or 2.

5. A method of knocking out a silver carp (Hypophthalmichthys molitrix) bmp6 gene, comprising annotating a first gRNA as set forth in SEQ ID No.1, a second gRNA as set forth in SEQ ID No.2, a third gRNA as set forth in SEQ ID No.3, and a Cas9 protein into fertilized eggs of the silver carp single cell stage.

6. The knockout method of claim 5, the annotated mass ratio of the first gRNA, the second gRNA, and the third gRNA is 1:1:1.

7. The knockout method according to claim 5 or 6, wherein mutants are selected from offspring of said silver carp.

8. The method of preparing a gRNA of claim 1 or 2, comprising:

designing and synthesizing primer pairs of gRNA;

carrying out PCR amplification on the primer pair;

Recovering the gRNA from the PCR amplified product.

9. The preparation method according to claim 8, wherein,

The primer pair for obtaining the first gRNA as shown in SEQ ID NO.1 comprises an upstream primer as shown in SEQ ID NO.4 and a downstream primer as shown in SEQ ID NO. 7;

the primer pair for obtaining the second gRNA as shown in SEQ ID NO.2 comprises an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO. 7;

the primer pair for obtaining the third gRNA described by SEQ ID NO.3 comprises an upstream primer described by SEQ ID NO.6 and a downstream primer described by SEQ ID NO. 7.

10. Use of the gRNA of claim 1 or 2 for breeding a silver carp mutant strain.