CN114717121A

CN114717121A - Fusarium genome large fragment knockout method

Info

Publication number: CN114717121A
Application number: CN202210410044.6A
Authority: CN
Inventors: 郭维; 唐婷婷
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2022-07-08

Abstract

The invention discloses a fusarium genome large fragment knockout method, which comprises the steps of determining a target large fragment to be knocked out and selecting a target site, simultaneously expressing the acquisition of CRISPR vectors of two sgRNAs, constructing and acquiring a donor vector, introducing the CRISPR vectors and the donor vector into fusarium, screening transformants step by step and extracting genome DNA, and designing a PCR detection primer and screening to obtain a large fragment knockout mutant; the method has simple technical composition and low cost, and can cause the damage of the thallus genome by shearing the DNA double strand, thereby improving the efficiency of homologous recombination, realizing the purpose of knocking out the large fragment of the Fusarium genome, and promoting the basic research, metabolic engineering and synthetic biology application of the Fusarium.

Description

Fusarium genome large fragment knockout method

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a Fusarium genome large fragment knockout method.

Background

The traditional filamentous fungus genetic transformation mainly realizes the directional modification of a genome by introducing a homologous fragment and then utilizing a DNA repair mechanism of thalli, and because the method does not introduce any DNA damage and directly utilizes a homologous recombination mechanism of thalli to repair, the efficiency is relatively low, and the method is limited to knock out smaller genes, a certain difficulty exists in knocking out large fragments on the genome, and the development of a new genome large fragment knock-out technology has important significance on the basic research, metabolic engineering, synthetic biology research and the like of filamentous fungi, so the invention provides a fusarium genome large fragment knock-out method to solve the problems in the prior art.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a method for knocking out large segments of fusarium genome, which has simple technical composition and low cost, and can improve the efficiency of homologous recombination by shearing double DNA strands to cause damage to the genome of a strain, thereby achieving the purpose of knocking out large segments of fusarium genome, and promoting basic research, metabolic engineering and synthetic biology application of fusarium.

In order to realize the purpose of the invention, the invention is realized by the following technical scheme: a Fusarium genome large fragment knockout method comprises the following steps:

step one, determining a large target segment to be knocked out, and selecting a target site 1 and a target site 2 at two ends of the large segment respectively;

step two, constructing two target site sequences on the same PFC332 carrier framework by a one-step cloning method to obtain a CRISPR carrier for simultaneously expressing two sgRNAs;

respectively selecting sequences without target sites on the left and right sides of the large fragment as homology arms, and constructing the sequences to two sides of the nourseothricin resistance gene to obtain a donor vector;

step four, introducing the CRISPR vector and the donor vector into fusarium through PFG-mediated protoplast transformation;

step five, screening the transformant step by utilizing hygromycin and nourseothricin to preliminarily obtain the transformant and extract genome DNA;

and step six, designing a PCR detection primer, carrying out PCR amplification by using the transformant genome DNA as a template, and screening to obtain a large fragment knockout mutant.

The further improvement lies in that: the length of the large fragment sequence in the first step is more than 15kb, and the fusarium in the fourth step is fusarium verticillioides but is not limited to the fusarium verticillioides.

The further improvement lies in that: the CRISPR vector for simultaneously expressing two sgRNAs in the second step is constructed by the following steps

S1, amplifying to obtain sgRNA1-1 and sgRNA1-2 fragments by using a PFC334 carrier as a template and PFC334_ PacI _ F/sgRNA1_ R, sgRNA1_ F/PFC334_ PacI _ R as a primer pair;

s2, performing fusion PCR amplification by using sgRNA1-1 and sgRNA1-2 as templates and PFC334_ PacI _ F/PFC334_ PacI _ R as a primer pair to obtain a sgRNA1 fragment;

s3, connecting a sgRNA1 fragment to a PFC332 vector PacI enzyme digestion site by a restriction enzyme PacI single enzyme digestion PFC332 vector through a one-step cloning method to obtain a PFC332_ sgRNA1 vector;

s4, amplifying to obtain sgRNA2-1 and sgRNA2-2 fragments by using a PFC334 carrier as a template and PFC334_ MauBI _ F/sgRNA2_ R, sgRNA2_ F/PFC334_ MauBI _ R as a primer pair;

s5, performing fusion PCR amplification by using sgRNA2-1 and sgRNA2-2 as templates and PFC334_ MauBI _ F/PFC334_ MauBI _ R as a primer pair to obtain a sgRNA2 fragment;

s6, a PFC332_ sgRNA1 vector is subjected to single enzyme digestion by a restriction endonuclease MauBI, and a sgRNA2 fragment is connected to a MauBI enzyme digestion site of a PFC332_ sgRNA1 vector in S3 by a one-step cloning method, so that a CRISPR vector PFC332_ sgRNA1+2 capable of expressing sgRNA1 and sgRNA2 simultaneously is obtained.

The further improvement lies in that: the nucleotide sequences of the primers PFC334_ PacI _ F in S1 and S2 are shown in SEQ ID NO. 1; the nucleotide sequence of the primer PFC334_ PacI _ R is shown in SEQ ID NO. 2; the nucleotide sequence of the primer sgRNA1_ F is shown in SEQ ID NO. 3; the nucleotide sequence of the primer sgRNA1_ R is shown in SEQ ID NO. 4; the sgRNA1-1 has a nucleotide sequence shown in SEQ ID NO. 27; the sgRNA1-2 has a nucleotide sequence shown in SEQ ID NO. 28; the sgRNA1 has the nucleotide sequence shown in SEQ ID NO. 29.

The further improvement lies in that: the nucleotide sequence of the primer PFC334_ MauBI _ F in the steps S4 and S5 is shown as SEQ ID NO. 5; the nucleotide sequence of the primer PFC334_ MauBI _ R is shown in SEQ ID NO. 6; the nucleotide sequence of the primer sgRNA2_ F is shown in SEQ ID NO. 7; the nucleotide sequence of the primer sgRNA2_ R is shown in SEQ ID NO. 8; the sgRNA2-1 has a nucleotide sequence shown as SEQ ID NO. 30; the sgRNA2-2 has a nucleotide sequence shown in SEQ ID NO. 31; the sgRNA2 has the nucleotide sequence shown in SEQ ID NO. 32.

The Fusarium genome large fragment knocking-out method is applied to gene large fragment knocking-out.

The invention has the beneficial effects that: the method has simple technical composition and low cost, and can cause the damage of the thallus genome by shearing the DNA double strand, thereby improving the efficiency of homologous recombination, realizing the purpose of knocking out the large fragment of the Fusarium genome, and promoting the basic research, metabolic engineering and synthetic biology application of the Fusarium.

Drawings

FIG. 1 is a flow chart of a method according to an embodiment of the present invention.

FIG. 2 is a flowchart illustrating a second application of the present invention.

Fig. 3 is a construction diagram of the CRISPR vector of the present invention.

FIG. 4 is a diagram showing the construction of the donor vector of the present invention.

FIG. 5 is a PCR assay of Fusarium genomic large fragment knockout mutants based on CRISPR/Cas9 technology of the invention.

Detailed Description

In order to further understand the present invention, the following detailed description will be made with reference to the following examples, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.

Example one

According to FIG. 1, the present embodiment provides a Fusarium genome large fragment knockout method, comprising the following steps:

step one, determining a target large fragment to be knocked out, wherein the sequence length of the target large fragment is larger than 15kb, and selecting a target site 1 and a target site 2 at two ends of the large fragment respectively;

the CRISPR vector for simultaneously expressing two sgRNAs is constructed by the following steps

the nucleotide sequence of the primer PFC334_ PacI _ F is shown in SEQ ID NO. 1; the nucleotide sequence of the primer PFC334_ PacI _ R is shown in SEQ ID NO. 2; the nucleotide sequence of the primer sgRNA1_ F is shown in SEQ ID NO. 3; the nucleotide sequence of the primer sgRNA1_ R is shown in SEQ ID NO. 4; the sgRNA1-1 has a nucleotide sequence shown in SEQ ID NO. 27; the sgRNA1-2 has a nucleotide sequence shown in SEQ ID NO. 28;

s2, performing fusion PCR amplification by using sgRNA1-1 and sgRNA1-2 as templates and PFC334_ PacI _ F/PFC334_ PacI _ R as a primer pair to obtain a sgRNA1 fragment; the sgRNA1 has a nucleotide sequence shown in SEQ ID NO. 29;

the nucleotide sequence of the primer PFC334_ MauBI _ F is shown in SEQ ID NO. 5; the nucleotide sequence of the primer PFC334_ MauBI _ R is shown in SEQ ID NO. 6; the nucleotide sequence of the primer sgRNA2_ F is shown in SEQ ID NO.7, and the nucleotide sequence of the primer sgRNA2_ R is shown in SEQ ID NO. 8; the sgRNA2-1 has a nucleotide sequence shown as SEQ ID NO. 30; the sgRNA2-2 has a nucleotide sequence shown in SEQ ID NO. 31;

s5, performing fusion PCR amplification by using sgRNA2-1 and sgRNA2-2 as templates and PFC334_ MauBI _ F/PFC334_ MauBI _ R as a primer pair to obtain a sgRNA2 fragment; the sgRNA2 has a nucleotide sequence shown in SEQ ID NO. 32;

s6, performing single enzyme digestion on a PFC332_ sgRNA1 vector by using a restriction endonuclease MauBI, and connecting a sgRNA2 fragment to a MauBI enzyme digestion site of a PFC332_ sgRNA1 vector in S3 by using a one-step cloning method to obtain a CRISPR vector PFC332_ sgRNA1+2 capable of simultaneously expressing sgRNA1 and sgRNA 2;

step four, introducing the CRISPR vector and the donor vector into Fusarium through PFG-mediated protoplast transformation, wherein the Fusarium is Fusarium verticillium but is not limited to Fusarium verticillium;

Example two

According to the illustration in fig. 2, the embodiment provides an application of a fusarium genome large fragment knockout method in gene large fragment knockout, firstly, according to an experimental purpose, a target large fragment to be knocked out is determined through bioinformatics analysis, two target sites are respectively designed at two ends of the large fragment, the two target sites are jointly constructed on the same PFC332 vector to form a CRISPR vector capable of releasing two sgrnas simultaneously, meanwhile, a homology arm is respectively designed at the left side and the right side of the large fragment and is respectively constructed at two sides of a nourseothricin gene to form a donor vector containing a large fragment knockout homology arm, the CRISPR vector and the donor vector are simultaneously introduced into a protoplast, and a large fragment knockout mutant is obtained through stepwise screening and PCR detection of hygromycin and nourseothricin. The specific experimental protocol is as follows:

determining large segments of a target

With the aim of researching the biosynthesis of Fusarium verticillium fumonisin, determining a 38604bp sequence in an FUM gene cluster as a target large fragment through bioinformatics analysis;

designing target sites

Selecting a 20bp sequence adjacent to the 5 ' end of the ' NGG ' PAM site from the left end of the target large fragment as a target site 1; on the right end of the target large fragment, a 20bp sequence close to the 5 ' end of the ' NGG ' PAM site is also selected as a target site 2; and (3) performing bioinformatics analysis on the two selected target sites, comparing the specificity of the two selected target sites in the fusarium verticillioides genome, and ensuring that the number of homologous bases of the two selected target sites and other sites cannot exceed 15 bp. The specific nucleotide sequence is as follows:

the nucleotide sequence of the target site 1 is shown as SEQ ID NO. 21;

the nucleotide sequence of the target site 2 is shown as SEQ ID NO. 22.

Construction of CRISPR vectors

Designing a primer according to the specific conditions of a target site and a carrier:

the nucleotide sequence of the primer PFC334_ PacI _ F is shown in SEQ ID NO. 1;

the nucleotide sequence of the primer PFC334_ PacI _ R is shown in SEQ ID NO. 2;

the nucleotide sequence of the primer FUM _ sgRNA1_ F is shown in SEQ ID NO. 23;

the nucleotide sequence of the primer FUM _ sgRNA1_ R is shown in SEQ ID NO. 24;

the nucleotide sequence of the primer PFC334_ MauBI _ F is shown in SEQ ID NO. 5;

the nucleotide sequence of the primer PFC334_ MauBI _ R is shown in SEQ ID NO. 6;

the nucleotide sequence of the primer FUM _ sgRNA2_ F is shown in SEQ ID NO. 25;

the nucleotide sequence of the primer FUM _ sgRNA2_ R is shown in SEQ ID NO. 26;

using a PFC334 carrier as a template and PFC334_ PacI _ F/FUM _ sgRNA1_ R, FUM _ sgRNA1_ F/PFC334_ PacI _ R as a primer pair, and performing PCR by using PrimeSTAR Max Premix (2X) to obtain FUM _ sgRNA1-1 and FUM _ sgRNA1-2 fragments through amplification; then, FUM _ sgRNA1-1 and FUM _ sgRNA1-2 are used as templates, PFC334_ PacI _ F/PFC334_ PacI _ R is used as a primer pair, fusion PCR is carried out by using PrimeSTAR Max Premix (2X), and a FUM _ sgRNA1 fragment is obtained through amplification; performing single enzyme digestion on a PFC332 vector by using a restriction enzyme PacI, and connecting a FUM _ sgRNA1 fragment to a PacI enzyme digestion site of the PFC332 vector by one-step cloning to obtain a PFC332_ FUM _ sgRNA1 vector;

taking a PFC334 carrier as a template, taking PFC334_ MauBI _ F/FUM _ sgRNA2_ R, FUM _ sgRNA2_ F/PFC334_ MauBI _ R as a primer pair, and carrying out PCR by using PrimeSTAR Max Premix (2X) to obtain FUM _ sgRNA2-1 and FUM _ sgRNA2-2 fragments through amplification; then, FUM _ sgRNA2-1 and FUM _ sgRNA2-2 are used as templates, PFC334_ MauBI _ F/PFC334_ MauBI _ R is used as a primer pair, and fusion PCR is carried out by using PrimeSTAR Max Premix (2X), so that a FUM _ sgRNA2 fragment is obtained through amplification; the vector PFC332_ FUM _ sgRNA1 is subjected to single enzyme digestion by a restriction enzyme MauBI, and a FUM _ sgRNA2 fragment is connected to the enzyme digestion site of the vector PFC332_ FUM _ sgRNA1 by one-step cloning, so that the vector PFC332_ FUM _ sgRNA1+2 for target large fragment knockout is obtained.

The nucleotide sequence of FUM _ sgRNA1-1 obtained by PCR amplification is shown in SEQ ID NO. 33; the nucleotide sequence of FUM _ sgRNA1-2 is shown in SEQ ID NO. 34; the nucleotide sequence of FUM _ sgRNA1 is shown in SEQ ID NO. 35; the nucleotide sequence of FUM _ sgRNA2-1 is shown in SEQ ID NO. 36; the nucleotide sequence of FUM _ sgRNA2-2 is shown in SEQ ID NO. 37; the nucleotide sequence of FUM _ sgRNA2 is shown in SEQ ID NO. 38.

Construction of the Donor vector

Selecting 3500bp sequences on the left side and the right side of a large fragment of the FUM gene cluster as homologous arms, and designing primers:

the nucleotide sequence of the primer L-SalI-F is shown as SEQ ID NO. 9;

the nucleotide sequence of the primer L-SalI-R is shown as SEQ ID NO. 10;

the nucleotide sequence of the primer R-EcoRI-F is shown in SEQ ID NO. 11;

the nucleotide sequence of the primer R-EcoRI-R is shown in SEQ ID NO. 12;

taking the genome DNA of fusarium verticillioides as a template, taking L-SalI-F/L-SalI-R as a primer pair, carrying out PCR by using PrimeSTAR Max Premix (2X), and amplifying to obtain an L-SalI fragment, wherein the obtained L-SalI nucleotide sequence is shown as SEQ ID NO. 39;

the pNrsR vector is singly cut by restriction endonuclease SalI, and an L-SalI fragment is connected to the SalI cutting site of the pNrsR vector through one-step cloning to obtain the pNrsR-L vector;

PCR is carried out by taking genome DNA of fusarium verticillioides as a template and R-EcoRI-F/R-EcoRI-R as a primer pair and using PrimeSTAR Max Premix (2X) to obtain an R-EcoRI fragment through amplification, and the obtained R-EcoRI nucleotide sequence is shown as SEQ ID NO. 40;

the method comprises the following steps of (1) singly cutting a pNrsR-L vector by restriction endonuclease EcoRI, connecting an R-EcoRI fragment to an EcoRI cutting site of the pNrsR-L vector through one-step cloning, and obtaining a pNrsR-L + R vector for target large fragment knockout;

PEG-mediated protoplast transformation

Fusarium verticillium was activated in advance on PDA plates and cultured at 25 ℃ for 7 days.

Collecting spores, placing in 150ml YEPD liquid culture medium, and shaking at 25 deg.C and 150rpm overnight for 10 h; collecting young plants, adding into 0.7MNaCl containing 0.1g of lysine enzymes and 0.05g of driselase to prepare 20ml of mixed solution, and shaking at 28 ℃ and 100rpm for 3h for enzymolysis; protoplasts were collected and adjusted to a concentration of 10⁸One per ml. Mixing 10 μ g PFC332_ FUM _ sgRNA1+2 vector and 10 μ g pNrsR-L + R vector, adding into 100 μ L protoplast, mixing, and standing on ice for 20 min; adding 100 μ l of 40% PEG3350 solution dropwise, mixing, and standing on ice for 30 min; adding 800 μ l FRB solution, shaking at 25 deg.C and 150rpm overnight for 12h to double wall; pouring the mixed solution after wall covering into an FRA plate containing hygromycin, and culturing at 25 ℃;

after a single transformant grows out, screening twice by using a PDA (personal digital assistant) plate containing nourseothricin, transferring the screened transformant to a nonreactive single PDA plate for amplification culture, preserving glycerol bacteria, and extracting genome DNA (deoxyribonucleic acid) from residual hyphae;

4 pairs of PCR detection primers are designed, and wild type Fusarium verticillium and transformant genome DNA are used as templates for amplification, and the mutant is obtained through identification.

The primer pair 1 is an internal primer of the FUM gene cluster and is used for detecting whether the FUM gene cluster still exists in a genome, and the nucleotide sequence of the specific primer is as follows:

the nucleotide sequence of the primer FUM-in-F is shown as SEQ ID NO. 13;

the nucleotide sequence of the primer FUM-in-R is shown as SEQ ID NO. 14.

The 2 nd pair of primers are internal primers of the nourseothricin screening marker and are used for detecting whether the nourseothricin screening marker enters the interior of a genome, and the nucleotide sequences of the specific primers are as follows:

the nucleotide sequence of the primer NrsR-in-F is shown as SEQ ID NO. 15;

the nucleotide sequence of the primer NrsR-in-R is shown as SEQ ID NO. 16.

The 3 rd pair of primers is a primer pair formed by building the front end of an upstream homologous arm and the interior of a nourseothricin screening marker and used for detecting whether a donor fragment correctly replaces the FUM gene cluster, and the specific primer nucleotide sequences are as follows:

the nucleotide sequence of the primer UP-F is shown as SEQ ID NO. 17;

the nucleotide sequence of the primer NrsR-R is shown as SEQ ID NO. 18.

The 4 th pair of primers is a primer pair built with the interior of the nourseothricin screening marker and the rear end of the downstream homology arm, and is used for detecting whether the doror fragment correctly replaces the FUM gene cluster, and the specific primer nucleotide sequences are as follows:

the nucleotide sequence of the primer NrsR-F is shown as SEQ ID NO. 19;

the nucleotide sequence of the primer DOWN-R is shown in SEQ ID NO. 20.

Through the steps, large fragment knockout mutants of the FUM gene cluster are obtained successfully, and a molecular biology technical basis is laid for functional research of the FUM gene cluster.

Target site sequence Listing

Target sites	Site nucleotide sequence	SEQ ID NO.
			1	5’-GGATCCATTGCCATGCGTCA-3’	21
2	5’-GGACCTTTCTATATCCGAGG-3’	22

Primer sequence Listing

The nucleotide sequence of the target site 1 is 'NNNNNNNNNNNNNNNNNNNN' in the nucleotide sequence of the sgRNA1_ F shown in the SEQ ID No.3, and 'NNNNNN' in the nucleotide sequence of the sgRNA1_ R shown in the SEQ ID No.4 is the first 6 bases of the target site 1.

The nucleotide sequence of target site 2 is 'nnnnnnnnnnnnnnnnnnnnnnnnnnnnn' in the nucleotide sequence of primer sgRNA2_ F shown in SEQ ID NO.7, and the nucleotide sequence of target site 2 is 'nnnnnnnnnnnnn' in the nucleotide sequence of primer sgRNA1_ R shown in SEQ ID NO. 8.

Figure 3 of the accompanying drawings is a construction diagram of a CRISPR vector, wherein

A is a fusion amplification graph of an sgRNA1 fragment and an sgRNA2 fragment, wherein 1 corresponds to an sgRNA1 fragment, and 2 corresponds to an sgRNA2 fragment;

b is a colony PCR screening chart constructed by a PFC332_ sgRNA1 vector, and 1/3/5/6/7 is a positive clone;

c is a colony PCR spot screening diagram constructed by a PFC332_ sgRNA1+2 vector, and 2/8 is a positive clone; m is 1kb DNA ladder.

FIG. 4 is a drawing of the construction of the donor vector, in which

A is a colony PCR screening chart constructed by a pNrsR-L vector, and 1-9 are positive clones;

b is a colony PCR screening picture constructed by a pNrsR-L + R vector, and 1/2/3/5/6/7 is a positive clone; m is 1kb DNA ladder.

FIG. 5 is a PCR assay of Fusarium genomic large fragment knockout mutants based on CRISPR/Cas9 technology, in which

A is a PCR screening picture of the 1 st pair of primers, which is a large-fragment inner primer;

b is a PCR screening chart of the 2 nd pair of primers, and is an internal primer of a screening marker nourseothricin resistance gene;

c is a PCR screening picture of the 3 rd pair of primers, which is a primer pair built by the front end of the upstream homologous arm and the interior of the nourseothricin screening marker;

d is a PCR screening chart of the 4 th pair of primers, and is a primer pair formed by building the interior of the nourseothricin screening marker and the rear end of a downstream homologous arm;

4 detection results are integrated, and 2-9/12-17 is a large fragment knockout mutant; 1: a wild-type control; 2-17: knocking out transformants from large fragments of FUM gene cluster; m is 1kb DNA ladder.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

SEQUENCE LISTING

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

<400> 28

tgaggacgaa acgagtaagc tcgtcnnnnn nnnnnnnnnn nnnnngtttt agagctagaa 60

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 180

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 240

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 300

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 360

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 420

cttggctcat taagacctca gccgagacag cag 453

<210> 29

<211> 993

<212> DNA

<213> sgRNA1

<220>

<221> misc_feature

<222> (523)..(528)

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

<220>

<221> misc_feature

<222> (566)..(585)

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

<400> 29

tagctgtttc cgctgagggt ttaatgcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgnnnnnnct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtcnnnnn nnnnnnnnnn nnnnngtttt agagctagaa 600

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 660

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 720

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 780

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 840

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 900

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 960

cttggctcat taagacctca gccgagacag cag 993

<210> 30

<211> 565

<212> DNA

<213> sgRNA2-1

<220>

<221> misc_feature

<222> (523)..(528)

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

<400> 30

cggggtctga cgctcagtgg aacgagcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgnnnnnnct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtc 565

<210> 31

<211> 453

<212> DNA

<213> sgRNA2-2

<220>

<221> misc_feature

<222> (26)..(45)

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

<400> 31

tgaggacgaa acgagtaagc tcgtcnnnnn nnnnnnnnnn nnnnngtttt agagctagaa 60

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 180

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 240

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 300

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 360

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 420

cttggctcta actcacgtta agggattttg gtc 453

<210> 32

<211> 993

<212> DNA

<213> sgRNA2

<220>

<221> misc_feature

<222> (523)..(528)

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

<220>

<221> misc_feature

<222> (566)..(585)

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

<400> 32

cggggtctga cgctcagtgg aacgagcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgnnnnnnct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtcnnnnn nnnnnnnnnn nnnnngtttt agagctagaa 600

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 660

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 720

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 780

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 840

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 900

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 960

cttggctcta actcacgtta agggattttg gtc 993

<210> 33

<211> 565

<212> DNA

<213> FUM_sgRNA1-1

<400> 33

tagctgtttc cgctgagggt ttaatgcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgggatccct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtc 565

<210> 34

<211> 453

<212> DNA

<213> FUM_sgRNA1-2

<400> 34

tgaggacgaa acgagtaagc tcgtcggatc cattgccatg cgtcagtttt agagctagaa 60

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 180

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 240

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 300

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 360

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 420

cttggctcat taagacctca gccgagacag cag 453

<210> 35

<211> 993

<212> DNA

<213> FUM_sgRNA1

<400> 35

tagctgtttc cgctgagggt ttaatgcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgggatccct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtcggatc cattgccatg cgtcagtttt agagctagaa 600

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 660

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 720

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 780

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 840

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 900

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 960

cttggctcat taagacctca gccgagacag cag 993

<210> 36

<211> 565

<212> DNA

<213> FUM_sgRNA2-1

<400> 36

cggggtctga cgctcagtgg aacgagcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgaggtccct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtc 565

<210> 37

<211> 453

<212> DNA

<213> FUM_sgRNA2-2

<400> 37

tgaggacgaa acgagtaagc tcgtcggacc tttctatatc cgagggtttt agagctagaa 60

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 180

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 240

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 300

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 360

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 420

cttggctcta actcacgtta agggattttggtc 453

<210> 38

<211> 993

<212> DNA

<213> FUM_sgRNA2

<400> 38

cggggtctga cgctcagtgg aacgagcgta agctccctaa ttggcccatc cggcatctgt 60

agggcgtcca aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc 120

tcaggagctg gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc 180

tctacactcg acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc 240

gcccggtgtg tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat 300

tttcctgctc tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat 360

attcatcttc ccatccaaga acctttattt cccctaagta agtactttgc tacatccata 420

ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 480

gcagcttgac taacagctac cccgcttgag cagacatcac cgaggtccct gatgagtccg 540

tgaggacgaa acgagtaagc tcgtcggacc tttctatatc cgagggtttt agagctagaa 600

atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 660

cttttggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca tgcttcggca 720

tggcgaatgg gactgattta atagctccat gtcaacaaga ataaaacgcg tttcgggttt 780

acctcttcca gatacagctc atctgcaatg cattaatgca ttggacctcg caaccctagt 840

acgcccttca ggctccggcg aagcagaaga atagcttagc agagtctatt ttcattttcg 900

ggagacgaga tcaagcagat caacggtcgt caagagacct acgagactga ggaatccgct 960

cttggctcta actcacgtta agggattttg gtc 993

<210> 39

<211> 3550

<212> DNA

<213> L-SalI

<400> 39

caagcttgca tgcctgcagg tcgactcaac ttttgcgatg gcggggtcca ttgtgtttgt 60

gacagataat cagattagcg caaattcgag acacaagcca cgatttagac gacgtcgtgg 120

cgcgtgtgag tcgtgtaagc gtcgtaaagt ccggtgtaag ttgcttggcc tcctccactg 180

ctttgtactg tacctagctt atggttctta tgactactag gtaatggcac aaacccttgc 240

aatcaatgcc aagtcagatt cccgtcaaga tggtacatag tcatctcagc ccgagctaac 300

cagaagagca gaaatcgtcg atagaatgct tatatagcag ctcaagctgg aaagctaacg 360

acggagatac agagcgtgga ccatcaggat cgccggtgca acatacaagg gggagtttga 420

cgcctcctca aacgagccca tatggtggaa tggcttgtaa tggatcagcg gaagagtctt 480

cgagatttga tggctcggat gcacacagcc ttttgatgag cctcagcaat actgactgta 540

cgactaatac tgaggcaaca gagatgtcgc agtggcagaa ttggttcgtt agcgacctac 600

ctctagggag cgatgtgcaa cttacaggga catctgatga gtctttgacc gactggttgg 660

acccccagct tgtcaaccca gcagataccg cggccattat gcaaaccctt tcgggggagg 720

atctatttaa atcatccggc ccaagttatg gcatgtctga gcctccatcg cgggacagtg 780

atcccggctt caacacggcg agaaatgaac aaatcgccgg ttttattcag aagcttcgct 840

cccagagacc attcgttttg cgcgatgaaa atcccgaggc accaccaggt aacatcaggg 900

gcttttatga tgcaaagttt atctctcaat gtttcgacgg tacgtaaggg tttaatcaca 960

cagcatatca ggtagaactg accttattcc cacccgaaag cgtgcgatgc agatccggaa 1020

gggtatatat atttcctcca agatttccgt actagcggca ctactctaac acaataacag 1080

tgttcgagta ttattggaga gaaaaagcat ggatttgatt gcagacgaga ttacaaaata 1140

tgccctggct gttgacacag aaacctctgt tttattccac tccttgatgg ctattggatg 1200

ccattctttg aatcttgacc aaggccatcg agcaattggc aaggggaaat atcctgcctc 1260

gaagtttttc aaagaagctt taaatgcgag acagcatttg cgggacagcc ctagtcttgg 1320

aggacttcag gtatgttgta catgtcgaga aactaaactc aaagtgatga gggattcctc 1380

aaactgattg tacgtactgt tgtaggctat tttgactatg gtaattccct caccagatat 1440

ctacctactt gctagtaagt tcttacacgc gcacaaggca tacttctccg ctcgagtcgg 1500

ggacgattcc acttcgagct tcctcgccga tgccgccttt tgcgtccaga cgttgcaact 1560

acacaacgcg ggtgcaattg aacagaaata caacagcttc ctagaacaac aagtggccaa 1620

acgtgccctt tggttcttat attcccttga gaagcctcgc tgcctcgccc aaggcttatt 1680

accggtttgt agcgctcgac attcccttat agcagcaagc agccttttct tgcctgtaca 1740

ctgtgtctga ttcttgcgac gcatagttga tccacgacga cttcgtcgac tacgatcctc 1800

cgccgtccgc aaactattcg actagtgagg ttgactggtt cgctatcaac gcccggtttg 1860

cgaggatctg ttcttctatc ataaaggagc gacttggatg caaattgggt cgcacaccat 1920

cacgacgcgg aaaagaccgt gcctctgagt atagcgcaag ctcggtaata aagaggcttg 1980

aatctctttt ggaggagtgg agagatgatc tcccctttgc cggcgatttt gacgcaaccc 2040

ggtctgacga atttgcggcg tcaacgtgtg cagagagacg tcacaggatt aagtgcctga 2100

acaaatactg gtccgccatc attgcaacac attcaggaca ggcgcgtggt gtagccgaag 2160

atggcggtgc aggtgtccga ttgagtaggg caagatgcat tgacgccgcc caagcaattt 2220

tgcagaatag ccactacgtc acctcgaccg acattttatc tgatatgtac gttctgggga 2280

gaccaagtgt gtcattatca actgcatagg ccatttcatc tttcctatcc tttcgatctc 2340

acttctgtac tgttgcctgc ttgtttatac taaccaagtg tttagctcac tttactacta 2400

catcaccgtt gcaacacggg tcatcatgac tgtggtgatc cgtgacagat ttgccagcga 2460

tatcacaggg gatcctgttc agaagaatac agccacacgc aaaagagaaa ctacgcatgg 2520

caggtcaata atgtcctatg tgggaattgc gattggtctc tttagtcgtc tttctttaga 2580

tatagacgtt cctgtagatg aggttactga gttgggaaaa ttaggaagat agataataaa 2640

acatatctat ccgtgtgttt tagccaactg tagttatata cgagagggtc caacttcact 2700

acgaccataa tccacccacc tctatgccag ccctgactca atggcacagc acgcacacca 2760

tcactaacgg taaactcaaa acgacagccg cttgcattga ggctaaagca cggttccggc 2820

ttatacgaac agaagtctga catctttcat gataagaaga agaagaagaa gctgtcggaa 2880

tgatacagac ccaaatcaga tatccgaccg gctcctggtc ccattccatt atgagccaat 2940

ggggcaattg ccccctttcc ctacccgagt gttttcggac gttactgtcc ctcacccggg 3000

aactctagca aactattggg atgtaggata ataatagtaa ccttcctact ggttgccgaa 3060

tttccgggtc tcttactcgt ctttgaggat actgacaatt gcgcttcgga gtattgcgtg 3120

tcctctcgga tgggagacta aatcgtatac aacaaccgga atttcgatgc tgattgcaat 3180

gatagagacg agacagatat ataaccatga aatacagtgt tattggagac tctgtatttg 3240

atccatcgca accttattat cgttttaaac gctactgata tcatcaacgt tcgacatggg 3300

agtaatcgaa tcaccctcaa gcaccacttc aggctctgcc gaagagatgg ctcaagccat 3360

caccggtcat gaggattcag ttttgcccgt tgctattgtt ggcatgggta tgaggctgcc 3420

gggtgggatc catactcctg atgaactttg gggcatgctt gtggagaagc gttccacccg 3480

atgtgagatc cccccaactc gattctcagt tgatgggttc cacaggattc tagtctagaa 3540

cagaagatga 3550

<210> 40

<211> 3550

<212> DNA

<213> R-EcoRI

<400> 40

tagaggatcc ccgggtaccg agctccaggt cggcatcgca ttcatagcac cgattgggct 60

tgtattgctc tccttcctgg gcgtctctat tgtggggaat tatgtcgggc tctatcaaaa 120

agcgtggatg ggcaaaatac agaaccgcgt ggctataact gccaacgtga tatccaacat 180

caaacacctc aaagtctccg ggatgactcg accagttgaa tccatcatcc aaaacactcg 240

agaatctgaa ctacgagcta gtcgtgggac tcgcaggctc cagattgcgt cgctcatcat 300

cgcctttgcg cccgatttga ctgcaccggg cattatgctc gcagccacca agtcgcagga 360

tttcagctct cataaggtct atactgcgat tgcccttctc acattgttga ctgtgccgct 420

tggcagtatc ttccgctctg tctcaccctt gatgtcggcg tttgcatgtc tagagcgtat 480

tcaagcattt cttgaacttg atacgcggaa ggaccctcgc ctgattacac attcgacgcc 540

agatacttca tcggcctcag gcgatgaaaa ggtctacgct gatccattgc ctatgtctag 600

aagctcagca gtcaaagtta ttcaagcaag ctttgggtgg cagggtaaag agcaggcatg 660

tttaaagaac atcaacttga ctgtcaacta ttctgccttg actgctatta ttgggcctgt 720

tggatcagga aagtcgacgt tgtgcaaagc cttgctcggt gaaacgccat tctcggctgg 780

caaggtcgtt ctggaacgcg atgctagctg taaagttggc tattgcgatc aaacaccctt 840

tatacgaaac tgcagcatca aggagaacat agttggcttc tctaaatgga acccagtccg 900

ctatcttgag gtggttgaag cgtcgatgct ctcatacgac ctcagggaat tacctgaggg 960

tgatgcaacg atagtgggaa gcggaggcat gactttgagc ggtggtcaaa aacagcgcat 1020

cgctattgct agatccctct atcttgacac aaggctcctg atattggatg atattctgag 1080

tggtcttgat actcagacgg aacaccatct tttccaacat gttcttagtc cgaatggcct 1140

cctgaagaag agagaaaacg cgccagccat tgtcttttcc actcattcgg tcaagtacgc 1200

ccggtgggct gatcacatct ttctactgaa tgaaaaaggc gagatgatcg agcaaggtag 1260

ctgggaggag ttgtcaacat atcaaagcca tttgcaaagc ctgtgcattc aggagaaagt 1320

acaaatgacg gatctgaagc agctcgaacc tgtggaaagc gagcagccac tggatgtcac 1380

tatgtcagag attgagcaaa ctcgttccag ccgccgtggt gctgataacc aagaaactat 1440

cgctagtggt gcggatagtt ctgcacgtca aaacggcgat cttggcgtct accgacacta 1500

ttttcgagct gttcctcctg tagccattat ttccttcgtc acgtcctcct tgtcctacgg 1560

attcctttac agctttccta acatttggtt aaaatggtgg ctattagacg ccgattcaac 1620

cagaccacat catcccaagg ccttttggaa tggtatttat gccatgttcc aaatactagc 1680

tcttctcagc gaactgctga ccatgtatct ggcgctcacc tacttcgctc tgatatctgg 1740

agcaacagtg cactcatcag cattgcgggc catcacacga gcaccactat acttctttgc 1800

cagtgtagac ctgggaacaa tcacgaatta tttctcacag gacatgactc tggtggatgg 1860

cgccctccct gcctcactaa ttcagtttgc tagcgatgta gcagcttctc ttgggatggc 1920

cggtaacctg gcagcgtcat ccccatacct agccgcctcg tatcctctct gctttttctt 1980

actttacttc gtcacaaagt actatttacg gacatctcgt cagcttcgcc tcctggatct 2040

tgaggccaag agtcctcttt agtaggttca tgtttgttgt tgactcatgt cgcacttgat 2100

ctgacttgaa tctcgcagtg cacacttcct cgagttggag aatggaatcg ctactatacg 2160

tgcagctgac tggacaggag aatatctcgt acaaagccgg ctgttgctcg acgtctccca 2220

acgccctgcc tatctcttgg ccatggttca acgctggctt ctatttattc tcaacacatt 2280

tgtttcgtta ctggccctct ttaccgttgc gcttgtcacg cagctcaaca atcacggaac 2340

agggttcgcc ggggcaggct taatatctct aatgcaaatt ggccagtttc tcaccaatgt 2400

cgtgagaagt tatgcgactc tggaagtttc aatgggtgcg gtcagtcgac taaaagctct 2460

gactgaaagc ccacatcgcg agtgtattga gggccaggag gttgtgccac ctcaggaatg 2520

gccttgcaga ggctccatca agattgatgg ggtttctgca tcctacgagt aagtctccca 2580

ccctcttcca agtctggcag tcggtacaat tctcacgccc taaacagcag ccaaaacgat 2640

caagtaaatg aaaagtcctt ttcactcagg gaattgaatc ttcatatcga ggctggtcag 2700

aaagtggcca tatgtggccg tacaggaagg tgtgtataac catcaagata tttacttggc 2760

atgcacaatc ctgtcgataa gaggtcatga actaagcaat cactaacata atgcttctca 2820

tattctagtg ggaaatcttc catcatacta cttcttcttc atatgctcag accgctgcgc 2880

aatactcgag aggatgctat caccatcgat ggcatttcca ttcaaaatgt tgatcccccg 2940

atcttgcgcg agagaatatt cgctgtgcca caagatacaa tattccttcc ccaaggctcg 3000

tcatggctgg agaatatgga gccttttgcg accaatgctg ctgagtgcag gtccgtctta 3060

gaggacgtca atctttggga tgtggttatt gcacaagggg gagatttgac cgcggctctg 3120

gactcagata ctcttagcca aggtcagagg cagctttttg gtctcgctcg tgctgtcttg 3180

agaaagagag caaaggcgca atccatgtca gaaccagcgc cccaaggtgg gcttctccta 3240

ctcgatgagc ctagttctgc tgtcgatttt gagacagaag ggttgatgca cagggttatc 3300

caacgtgagt tctgcgagta cacggtcatc atggttactc acagacttga atttatcact 3360

caaatacaca gcgtagggca ggttagcgta gatacccagc aaggactttt cgaccgggtg 3420

cttgtggttg atgccgggac tatagttgaa gatggccatc cagctcagct tctggaatcg 3480

aaagagggaa aatttagagc actctgggag gcgtcaagag tctaagaatt cgtaatcatg 3540

gtcatagctg 3550

Claims

1. A Fusarium genome large fragment knockout method is characterized by comprising the following steps:

2. The method of claim 1, wherein the knockout of the large fragment of fusarium genome is performed by: the length of the large fragment sequence in the first step is more than 15kb, and the fusarium in the fourth step is fusarium verticillioides but is not limited to the fusarium verticillioides.

3. The method of claim 1, wherein the knockout of the large fragment of fusarium genome is performed by: the CRISPR vector for simultaneously expressing two sgRNAs in the second step is constructed by the following steps

s3, performing single enzyme digestion on the PFC332 vector by using a restriction enzyme PacI, and connecting a sgRNA1 fragment to the PacI enzyme digestion site of the PFC332 vector by using a one-step cloning method to obtain a PFC332_ sgRNA1 vector;

4. The method of claim 3, wherein the knockout of the large genomic fragment of Fusarium is carried out by: the nucleotide sequences of the primers PFC334_ PacI _ F in S1 and S2 are shown in SEQ ID NO. 1; the nucleotide sequence of the primer PFC334_ PacI _ R is shown in SEQ ID NO. 2; the nucleotide sequence of the primer sgRNA1_ F is shown in SEQ ID NO. 3; the nucleotide sequence of the primer sgRNA1_ R is shown in SEQ ID NO. 4; the sgRNA1-1 has a nucleotide sequence shown in SEQ ID NO. 27; the sgRNA1-2 has a nucleotide sequence shown in SEQ ID NO. 28; the sgRNA1 has the nucleotide sequence shown in SEQ ID NO. 29.

5. The method of claim 3, wherein the knockout of the large fragment of Fusarium genome is carried out by: the nucleotide sequence of the primer PFC334_ MauBI _ F in the steps S4 and S5 is shown as SEQ ID NO. 5; the nucleotide sequence of the primer PFC334_ MauBI _ R is shown in SEQ ID NO. 6; the nucleotide sequence of the primer sgRNA2_ F is shown as SEQ ID NO. 7; the nucleotide sequence of the primer sgRNA2_ R is shown in SEQ ID NO. 8; the sgRNA2-1 has a nucleotide sequence shown as SEQ ID NO. 30; the sgRNA2-2 has a nucleotide sequence shown in SEQ ID NO. 31; the sgRNA2 has the nucleotide sequence shown in SEQ ID NO. 32.

6. Use of the Fusarium genomic large fragment knock-out method of any one of claims 1-5 for large fragment knock-out of genes.