CN112375781B - Application of pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing - Google Patents

Abstract

The invention relates to an application of a pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing, belonging to the technical field of plant genetic engineering. Including the selection of PagPDS gene target sites; designing gRNA; construction of a CRISPR/Cas9 gene knockout vector; genetic transformation of poplar; obtaining and counting transgenic plants; the CRISPR system provides directed editing of poplar genes. The pC1300-MAS-Cas9 gene editing system is simple, quick and efficient in application in 84K poplar gene editing, and a multi-gene editing system is constructed by applying homotail enzyme preparation, and can be used for editing an unlimited number of target genes simultaneously in theory by taking pC1300-Cas9 and SK-gRNA as skeleton carriers. The invention also optimizes the promoter, drives Cas9 with MAS promoter, and studies its editing efficiency in 84K poplar.

Description

Application of pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing

Technical Field

The invention relates to application of a CRISPR/Cas9 gene editing system in woody plant gene editing, in particular to application of a pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing, namely application of a MAS promoter-driven Cas9 gene editing system in 84K poplar gene editing, and belongs to the technical field of biological gene engineering.

Background

The type II CRISPR/Cas9 system is an acquired immune system from streptococcus that is able to resist invasion by foreign genes. Through artificial modification, the system is widely applied to animals and plants. It consists essentially of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and a specific Cas9 protein. CRISPR is a clustered regularly interspaced short palindromic DNA repeat consisting of a series of short highly conserved forward repeats and a sequence spacer arrangement of similar length. The Cas9 protein is a multi-domain protein consisting of 1409 amino acids, containing two nuclease domains RuvC-like and HNH. The CRISPR/Cas9 system was first successfully used in 2013 for gene editing of eukaryotic cells by Seung et al (Seung Woo Cho et al, 2013). The CRISPR/Cas 9-based gene editing system comprises two parts: sgRNA and enzyme Cas9. This system is capable of specifically recognizing a target sequence adjacent to a PAM (NGG) motif and cleaving 3nt upstream thereof, while the RuvC-like domain cleaves 3-8 nt upstream of the PAM region to form a DSB (double strains break double strand break), and then the organism repairs damaged DNA by its own repair mechanism such as homologous recombination, non-homologous recombination, and thus Indel (including deletion and insertion) phenomenon. CRISPR/Cas9 vector construction systems for application on plants include vector construction systems based on gene gun transformation and vector construction systems based on agrobacterium transformation. In the aspect of agrobacterium tumefaciens transformation commonly used at present, the construction process of a plant gene knockout carrier system based on a CRISPR/Cas9 technology is complicated, and multiple steps are needed to integrate each part onto a binary carrier.

Poplar is a model plant of woody plant research, also an important source of paper, wood, building materials, and biofuels, with high economic and ecological value (Jansson and Douglas, 2007). '84K' hybrid poplar (Populus alba x P.glandulosa) is widely planted in North China and is also an ideal research material for vascular cambium activity and wood formation by many research institutions. The '84K' hybrid poplar genomic sequence has been published (Huanget al., 2019), with a clear genetic background and high genetic conversion (Liet., 2017). Therefore, we select '84K' hybrid poplar for technical research of construction of gene editing vector and optimization of editing efficiency.

Gene editing techniques based on CRISPR/CAS9 have been applied to gene editing of woody plants. Researchers at University of Georgia (UGA) use CRISPR/Cas9 gene editing tools for the first time, and knock out key enzyme genes for lignin synthesis such as 4CL genes through gene editing, so that lignin content in wood can be effectively reduced (Zhou, jacobs et al 2015). Four guide RNAs (grnas) were designed using CRISPR/Cas9 system at university of southwest Luo Keming, targeting different genomic sites of aspen phytoene dehydrogenase gene 8 (PtoPDS), effectively editing genomic sequences in woody plants (Fan, liu et al 2015). Due to the high complexity of the genome of poplar, the genome editing efficiency of current CRISPR/CAS9 vectors in different local varieties of poplar is generally low. There is a need to modify the backbone vector to optimize the gene editing efficiency to increase the gene editing efficiency of Cas9 in poplar genomes.

The expression of the key nuclease gene Cas9 of the CRISPR/Cas9 gene editing vector is usually driven by a cauliflower mosaic virus 35S promoter (CaMV 35S) promoter or ubiquitin promoter with higher promoter activity, and researches show that: the activity of the promoter affects Cas9 expression and the final gene editing efficiency. Currently, in CRISPR/Cas9 vectors for poplar genome editing, the promoters of Cas9 genes are mostly cauliflower mosaic virus 35S promoter (CaMV 35S) promoters. Promoter-driven Cas9 gene expression vectors for high activity in poplar edited for poplar genome have not been reported. Therefore, there is a need to construct a high promoter activity driven expression CRISPR/CAS9 gene expression backbone vector in poplar, and to improve the efficiency of targeted gene editing.

Therefore, the pC1300-MAS-Cas9 gene editing system driven by the high-activity MAS promoter is applied to woody plant 84K poplar gene editing, so that plants with multiple target mutations can be conveniently and efficiently generated, and the technical problem to be solved in the technical field is urgently solved.

Disclosure of Invention

The invention aims to provide a simple and effective application of a pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing, aiming at the defects existing in the prior art.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the application of the pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing comprises the following steps:

step (1): selection of PagPDS Gene target sites

According to the 84K Yang Jiyin sequence, a CDS region of the PagPDS gene is found, the structure is analyzed, a potential Cas9 target site is searched, and gRNA is designed according to the position and GC content of the target site;

step (2): design of gRNA

Synthesizing a pair of DNA primers with complementary sequences, pagPDS_F and PagPDS_R according to the selected gRNA template sequence, respectively referring to sequences 3 and 4 in a sequence table, and synthesizing oligonucleotides;

according to the design principle of PAM sequences, 3 gRNAs, namely gRNA1, gRNA2 and gRNA3, are designed in sequences without SNP loci, and see sequences 9-11 in a sequence table;

step (3): construction of CRISPR/Cas9 Gene knockout vector

1) Annealing polymerization of oligonucleotides

Designing a desalting oligonucleotide chain (oligo) with a specific linker according to the subsequently ligated carrier linker sequence (g++ is F-direction; g- -R-direction), and diluting the synthesized oligonucleotide primer with ddH20 water (final concentration 100. Mu.M); annealing reaction system: 1 mu L of sense oligonucleotide (100 mu M), 1 mu L of antisense oligonucleotide (100 mu M), adding ddH20 to complement to 50 mu L, uniformly mixing the prepared annealing reaction buffer solution, centrifuging briefly, then using a PCR instrument, standing at 95 ℃ for 5min at room temperature, deforming and annealing after mixing the primers to form an oligonucleotide fragment with a sticky end, and immediately using the oligonucleotide fragment after standing at room temperature or storing the oligonucleotide fragment at-20 ℃ for a long time;

2) Construction of a Single intermediate vector

Inserting the gRNA sequence into a vector containing SK-gRNA (the sequence of which is shown as a sequence 12 in a sequence table);

3) Multiple intermediate support polymerization

Polymerizing an infinite number of SK-gRNA intermediate vectors by utilizing the property that BamHI and BglII are isotail enzymes, and carrying out enzyme digestion on the vectors by KpnI and BamHI; the fragment was digested with Spel/Xba I, xho I/Sal I, and BamH I/BgI II;

4) Construction to final vector

The pC1300-Cas9 vector is digested with KpnI and BamHI, SK-gRNA is digested with KpnI and BglII and fragments are recovered and ligated to the pC1300-Cas9 vector using a final vector ligation system;

5) Transformation competence

After transformation, 100 μl of coated plates (LB+Kan) were taken, 10 single clones were picked on LB+Kan plates the next day, 1.5mL centrifuge tubes were added with LB+Kan, shaking was performed in a shaker at 37℃at 250rmp, and after shaking, PCR detection and sequencing was performed;

firstly, gRNA1, gRNA2 and gRNA3 are connected into SK-gRNA through AarI enzyme, spel/XbaI, xhoI/SalI and BamHI/BgI II enzyme cutting sites are carried out in SK-gRNA1, SK-gRNA2 and SK-gRNA3 vectors, through homotail enzyme digestion (SK-gRNA 1 is cut by KpnI+SalI, SK-gRNA2 is cut by XhoI+XbaI, SK-gRNA3 is cut by Spel+BgI II) and recovered, and meanwhile, the final vector pC1300-Cas9 is cut and recovered by KpnI and BamHI; three intermediate vectors are simultaneously connected to a final vector pC1300-2x35S-Cas9/pC1300-MAS-Cas9 with KpnI and BamH I cleavage sites, which are respectively started by 2x35S and MAS promoters;

step (4): genetic transformation of poplar;

step (5): acquisition and statistics of transgenic plants

Respectively obtaining 40 PagPDS-2x35S-Cas9 and PagPDS-MAS-Cas9 transgenic plants by an agrobacterium transformation leaf disc method, wherein the transgenic plants of the two vectors have albino phenotype; among the 40 transgenic plants in PagPDS-2x35S-Cas9, albino seedlings are 27, the mutation rate is 67.5%, wherein 21 plants (77.8%) are of a pure-whitening phenotype, and 6 plants (22.3%) are of a yellowish green phenotype; in PagPDS-MAS-Cas9, the albino seedlings are 30 in 40 transgenic plants, the mutation rate is 75%, wherein 23 plants (76.7%) are pure white standard type, and 7 plants (23.3%) are yellow-green phenotype;

step (6): directional editing of poplar genes by CRISPR system

1) Identification of transgenic plant DNA level

After the leaves are subjected to agrobacteria dip-dyeing, screening and growing in a screening culture medium containing Hyg, and identifying rooting resistant seedlings after the material is subjected to differentiation, elongation and rooting processes;

2) Mutation identification of transgenic plants

After obtaining positive transgenic plants, using specific primers for gRNA target site detection, S_PDS_F and S_PDS_R, the sequences of which are respectively shown as sequences 7 and 8 in a sequence table, carrying out PCR amplification to obtain DNA sequence fragments with potential target site modification, then connecting the fragments to a T carrier after electrophoretic separation, transforming escherichia coli to obtain single colonies, extracting plasmids, sequencing the target sites, comparing the sequencing results with a control sequence, and analyzing mutation events.

Preferably, in the step (1), the PagPDS gene has a PagPDS form P.glandulosa sequence shown in a sequence table and a sequence 1,PagPDS form P.alba sequence shown in a sequence table and a sequence 2.

Preferably, in the step (2), after selecting the gRNA, it is further required to perform sequence verification on the gRNA target site on the target material gene, whether the gRNA exists in the target plant, then, in order to prevent the gRNA from being off-target due to mutation of nucleotides, extracting the gene DNA of the target poplar, referring to the poplar gene sequence, designing primers at 150-200bp upstream and downstream of the target site respectively, performing clone amplification on the target strip containing the target gene, detecting whether the target site sequence in the target plant is consistent with the sequence of the populus trichocarpus, and if not, performing appropriate modification on the gRNA according to the sequencing to obtain the oligonucleotide primer: pagPDS_F and PagPDS_R have sequences respectively shown as sequence 3 and sequence 4 in the sequence table.

Preferably, step (3) 2) is specifically as follows: firstly, carrying out Aar I digestion on SK-gRNA, wherein an Aar I digestion SK-gRNA carrier system is shown in the following table 1 to form a carrier with a sticky end, and then, connecting the carrier with the sticky end with the oligonucleotide fragment to convert DH5 alpha to obtain a connected final carrier; the common primer T3 and g-are matched to carry out colony PCR positive detection, and then the common primer T7 or T3 is used to carry out sequencing detection to verify whether the colony PCR positive detection is correct;

TABLE 1AarI cleavage SK-gRNA vector System

Enzyme digestion is carried out at 37 ℃ for 3-6hr, and the enzyme digestion product is purified by the kit.

Preferably, in step (3), 4), the final loaded ligation system is as shown in Table 2 below.

Table 2 end-load connection system

Preferably, in step (3) 5), the specific steps are as follows: firstly, carrying out Aar I enzyme digestion on SK-gRNA to form a carrier with a sticky end, carrying out denaturation annealing after mixing the gRNA to form a fragment with the sticky end, connecting the carrier and the fragment, converting DH5 alpha to obtain a connecting plasmid, and sequencing;

polymerizing 3 SK-gRNA intermediate vectors by utilizing the property that BamHI and BglII are isotail enzymes, performing enzyme digestion by using KpnI and BamHI as vectors to provide segmented SK-gRNA1, and performing enzyme digestion by using KpnI+SalI; SK-gRNA2 was digested with XhoI+XbaI; SK-gRNA3 was digested with Spel+BgI II.

Preferably, step (4) is specifically as follows:

1) Explants as transformation material: taking stem segments and tender leaf blades of 84 poplar growing for 3-4 weeks as explant transformation materials, cleaning the blades with clean water after taking materials, sterilizing in an ultra clean bench with 20% sodium hypochlorite for 20min, cleaning with sterile distilled water for at least 5 times, ensuring that no sodium hypochlorite remains on the surface of the material, and sucking redundant distilled water with sterile filter paper;

2) Tissue culture seedlings are used as transformation materials: cutting off plant leaves in an ultra-clean bench by scissors, taking small discs with obvious main veins by using a sterile 6mm puncher, and putting the small discs into a normal culture medium for standby so as to prevent water dispersion;

3) Taking out the stored carrier agrobacterium bacterial liquid in an ultralow temperature refrigerator at the temperature of 80 ℃ below zero, placing the carrier agrobacterium bacterial liquid on ice for melting, using a sterilized inoculating needle, taking a small amount of bacterial liquid, scraping the bacterial liquid on a prepared YEB solid culture medium containing 100mg/L Kan and 50mg/LRif, sealing the solid culture medium, inversely placing the solid culture medium in a 28 ℃ incubator for culturing for 48 hours, after single colony grows out, picking up the single colony, shaking the bacterial liquid in LB liquid culture medium (200 mL) containing 100mg/L Kan and 50mg/L Rif overnight (shaking table at the temperature of 28 ℃), and keeping the bacterial liquid to expand in the logarithmic phase;

4) Pouring the bacterial liquid into a sterilized centrifugal bottle, centrifuging for 30min (2560 rpm), discarding the upper bacterial liquid, and suspending the precipitate (OD 600 = 0.3-0.4) with the prepared suspension;

5) Culturing the suspended bacterial liquid in a shaking table with the temperature of 50-1000rmp for 1h;

6) The explant or tissue culture material (with or without 2-7d preculture) is soaked in the suspended bacterial liquid for dip dyeing: shaking in a 50rmp shaking table for 1-2h at room temperature;

7) After the dip dyeing is finished, sucking redundant dip dyeing liquid into the leaf by using sterile filter paper, transferring the leaf into a differentiation culture medium, and culturing in a dark inversion mode for 2d, wherein the optimal temperature is 19-21 ℃;

8) After two days, the agrobacterium grows out of the periphery of the dip-dyed material, the material is required to be cleaned, the material is cleaned by sterile water for four times, the filtered washing liquid is used for cleaning once, and after the washing liquid is added, the material is placed in a shaking table with 50-1000rmp for cleaning for 1h;

9) Placing the cleaned material in a differentiation medium containing 500 mg/Lcefotam and 2mg/L Hyg of antibiotics, culturing in dark for 2 weeks, and culturing under light for two weeks;

10 After callus growth, transferring the material to an elongation culture medium containing 50mg/L Hyg, and replacing the culture medium every 2-4 weeks;

11 When the material height is 2cm, transferring the material into rooting culture medium containing 2mg/L Hyg, and the whole transformation process is 3-8 months.

Preferably, step (6) 1) is specifically as follows: taking a small number of leaves of a rooting plant screened by Hyg, extracting DNA (deoxyribonucleic acid) for PCR (polymerase chain reaction) detection, taking a PCR amplification product with plasmid as a template as a positive control, taking a PCR amplification reaction product with unconverted wild plant gene DNA as a template as a negative control, taking a PCR reaction system as 20 mu L, uniformly mixing various components, and carrying out PCR reaction, wherein the PCR reaction procedure is as follows: after pre-denaturation at 98 ℃ for 5min, 30 cycles are carried out, wherein each cycle is denaturation at 98 ℃ for 5s, annealing at 62 ℃ for 5s and extension at 72 ℃ for 20s, finally extension at 72 ℃ is carried out for 1min, PCR products are detected by 1.0% agarose gel electrophoresis, and the observation results show that PCR detection primers are shown as sequences 5 and 6 in a sequence table; the target band 396bp.

Preferably, step (6) 2) is specifically as follows:

sanger sequencing was performed on the transgenic plant extract DNA, pagPDS-2x35S-Cas9 at the T1 site, and 53.75% of the mutations in all albino phenotype plants were single nucleotide insertions, and the rate of detection of at least one allelic mutation was 65% in 26 edited plants; 21 strains with simultaneous mutations of two alleles, including 20 homozygous mutations and 1 biallelic mutation; in the T2/T3 locus, the mutation rate was 67.5%, including 21 biallelic mutations and 4 homozygous mutations, and a deletion of 2 to 5nt was found near PAM;

in the PagPDS-MAS-Cas9 system, 30 plants were albino phenotype, mutation rate was 75%, pure-whitened plants 23 (76.7%), yellow-green phenotype plants 7 (23.3%); albino phenotype plants had alleles with significant large fragment deletions at T1 and T2/T3; regarding analysis of T1, mutations of at least one allele were detected in 29 plants (72.5%), with 22 mutations in both alleles, including 19 homozygous mutations and 3 bi-allelic mutations, and a mutation rate of 72.5% at the T2/T3 locus, including 20 homozygous mutations and 2 bi-allelic mutations.

The beneficial effects are that:

the pC1300-MAS-Cas9 gene editing system is simple, quick and efficient in application of 84K poplar gene editing, utilizes homotail enzyme preparation, constructs a multi-gene editing system, takes pC1300-MAS-Cas9 and SK-gRNA (AtU 6-26 driven intermediate vector) as a skeleton vector, and can be used for editing an unlimited number of target genes simultaneously in theory; the invention also optimizes the promoter, drives Cas9 by using the MAS promoter, and defines the editing efficiency in 84K poplar.

The invention is further described below with reference to the drawings and the detailed description, but is not meant to limit the scope of the invention.

Drawings

FIG. 1 is a diagram showing alignment of PagPDS gene in example 1 of the present invention.

FIG. 2 is a schematic representation of PagPDS target sites (T1-T3) and their sequences in example 1 of the invention.

FIG. 3 is a schematic diagram of a skeletal support of example 1 of this invention.

FIG. 4 is a schematic diagram of the vector construction procedure of example 1 of the present invention.

FIG. 5 is a schematic structural diagram of the final vector of example 1 of the present invention.

FIG. 6 is a phenotypic map of PagPDS-2x35s/MAS-Cas9 mediated transgenic plants in example 1 of the invention.

FIG. 7 is a comparison of the sequencing result of the target site in example 1 of the present invention with a control sequence.

Detailed Description

Unless otherwise specified, in the examples of the present invention, the raw materials used are all conventional raw materials commercially available in the art; the preparation method and the detection method are all conventional methods in the technical field.

Example 1

The application of the pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing comprises the following steps:

step (1): selection of PagPDS Gene target sites

According to the 84K Yang Jiyin sequence, finding the CDS region of the PagPDS gene and analyzing the structure, designing primers according to the known sequence, cloning and sequencing the PDS gene in the 84K poplar, searching potential Cas9 target sites (san der et al., 2010) by utilizing an online analysis platform CRISPR-P (http:// cbi.hzau.edu.cn/cgi-bin/CRISPR) and ZiFiT Targeted version4.2 (http:// zi t.components. Org/ZiFiT/introduction. Aspx), and designing gRNA according to the position and GC content of the required target sites;

cloning PDS gene (1-3 exons) in 84K poplar, and comparing the sequences in FIG. 1, wherein the sequence is shown in FIG. 1 and is the PagPDS gene sequence comparison chart in the example 1 of the invention; blue lines represent exons; red lines indicate PAM sequences; yellow highlighting gRNA sequence; wherein, pagPDS form P.glandulosa sequence is shown in sequence 1,PagPDS form P.alba in the sequence table and sequence 2 in the sequence table;

step (2): design of gRNA

Synthesizing a pair of DNA primers with complementary sequences, pagPDS_F and PagPDS_R according to the selected gRNA template sequence, wherein the synthetic oligonucleotides are required to be PAGE purified oligonucleotides respectively referring to sequences 3 and 4 in a sequence table;

after selecting the gRNA, the sequence verification is also required to be carried out on the target locus of the gRNA on the target material gene, whether the gRNA exists in the target plant or not, then, in order to prevent the gRNA from being off target due to mutation of nucleotide, the gene DNA of the target poplar is extracted, the gene sequence of the target poplar is referenced, primers are respectively designed at the upstream and downstream 150-200bp of the target locus, the cloning amplification is carried out on the target locus containing the target gene, whether the sequence of the target locus in the target plant is consistent with the sequence of the populus trichocarpus is detected, if not, the gRNA is properly modified according to the sequencing standard, and the oligonucleotide primer is obtained: pagPDS_F and PagPDS_R, the sequences of which are respectively shown as sequence 3 and sequence 4 in the sequence table;

according to the design principle of PAM sequence, 3 gRNAs, namely gRNA1, gRNA2 and gRNA3 (see sequences 9-11 in a sequence table respectively), are designed in the sequence without SNP locus, as shown in figure 2, which is the PagPDS target locus (T1-T3) and the sequence schematic diagram thereof in the embodiment 1 of the invention; wherein PAM (5 '-NGG-3') is represented by red; blue represents exons (CDS 1-CDS 4); orange represents introns;

step (3): construction of CRISPR/Cas9 Gene knockout vector

1) Annealing polymerization of oligonucleotides

Designing a desalting oligonucleotide chain (oligo) with a specific linker according to the subsequently ligated carrier linker sequence (g++ is F-direction; g- -R-direction), and diluting the synthesized oligonucleotide primer with ddH20 water (final concentration 100. Mu.M); annealing reaction system: 1 mu L of sense oligonucleotide (100 mu M), 1 mu L of antisense oligonucleotide (100 mu M), adding ddH20 to complement to 50 mu L, uniformly mixing the prepared annealing reaction buffer solution, centrifuging briefly, then using a PCR instrument, standing at 95 ℃ for 5min at room temperature, deforming and annealing after mixing the primers to form an oligonucleotide fragment with a sticky end, and immediately using the oligonucleotide fragment after standing at room temperature or storing the oligonucleotide fragment at-20 ℃ for a long time;

2) Construction of a Single intermediate vector

Inserting the gRNA sequence into a vector containing SK-gRNA (the sequence of which is shown as a sequence 12 in a sequence table): firstly, carrying out Aar I digestion on SK-gRNA, wherein an Aar I digestion SK-gRNA carrier system is shown in the following table 1 to form a carrier with a sticky end, and then, connecting the carrier with the sticky end with the oligonucleotide fragment to convert DH5 alpha to obtain a connected final carrier; the common primer T3 and g-are matched to carry out colony PCR positive detection, and then the common primer T7 or T3 is used to carry out sequencing detection to verify whether the colony PCR positive detection is correct;

TABLE 1AarI cleavage SK-gRNA vector System

Enzyme cutting at 37deg.C for 3-6hr, and purifying enzyme-cut product with kit;

3) Multiple intermediate support polymerization

Polymerizing an infinite number of SK-gRNA intermediate vectors by utilizing the property that BamHI and BglII are isotail enzymes, and carrying out enzyme digestion on the vectors by KpnI and BamHI; the fragment was digested with Spel/Xba I, xho I/Sal I, and BamH I/BgI II;

4) Construction to final vector

The pC1300-Cas9 vector is subjected to enzyme digestion by KpnI and BamHI, SK-gRNA is subjected to enzyme digestion by KpnI and BglII, fragments are recovered, and are connected to the pC1300-Cas9 vector, and a final-load connection system is shown in the following table 2, wherein the sequences of gRNA1, gRNA2 and gRNA3 are respectively shown in sequences 9-11 in a sequence table;

table 2 end-load connection system

5) Transformation competence

After transformation, 100 μl of coated plates (LB+Kan) were taken, 10 single clones were picked on LB+Kan plates the next day, 1.5mL centrifuge tubes were added with LB+Kan, shaking was performed in a shaker at 37℃at 250rmp, and after shaking, PCR detection and sequencing was performed;

FIG. 3 is a schematic diagram of a skeletal support of example 1 of the present invention; the vector consists of a skeleton vector SK-gRNA and pC1300-Cas 9;

firstly, gRNA1, gRNA2 and gRNA3 are connected into SK-gRNA through AarI enzyme, spel/XbaI, xhoI/SalI and BamHI/BgI II enzyme cutting sites are carried out in SK-gRNA1, SK-gRNA2 and SK-gRNA3 vectors, through homotail enzyme digestion (SK-gRNA 1 is cut by KpnI+SalI, SK-gRNA2 is cut by XhoI+XbaI, SK-gRNA3 is cut by Spel+BgI II) and recovered, and meanwhile, the final vector pC1300-Cas9 is cut and recovered by KpnI and BamHI; three intermediate vectors were simultaneously ligated to final vector pC1300-Cas9 with KpnI and BamH I cleavage sites, which were started by the 2x35S and MAS promoters, respectively;

FIG. 4 is a schematic diagram showing the steps of constructing a vector according to example 1 of the present invention: firstly, carrying out Aar I enzyme digestion on SK-gRNA to form a carrier with a sticky end, carrying out denaturation annealing after mixing the gRNA to form a fragment with the sticky end, connecting the carrier and the fragment, converting DH5 alpha to obtain a connecting plasmid, and sequencing;

FIG. 5 is a schematic structural diagram of the final vector of example 1 of the present invention: polymerizing 3 SK-gRNA intermediate vectors by utilizing the property that BamHI and BglII are isotail enzymes, performing enzyme digestion by using KpnI and BamHI as vectors to provide segmented SK-gRNA1, and performing enzyme digestion by using KpnI+SalI; SK-gRNA2 was digested with XhoI+XbaI; SK-gRNA3 was digested with Spel+BgI II;

the sequence of the final vector pC1300-MAS-Cas9-PagPDS constructed finally is shown as a sequence 13 in a sequence table; the sequence of the final vector pC1300-2X35S-Cas9-PagPDS constructed finally is shown as sequence 14 in the sequence table.

Step (4): genetic transformation of poplar

The conversion steps are as follows:

1) Explants as transformation material: taking stem segments and tender leaf blades of 84 poplar growing for 3-4 weeks as explant transformation materials, cleaning the blades with clean water after taking materials, sterilizing in an ultra clean bench with 20% sodium hypochlorite for 20min, cleaning with sterile distilled water for at least 5 times, ensuring that no sodium hypochlorite remains on the surface of the material, and sucking redundant distilled water with sterile filter paper;

2) Tissue culture seedlings are used as transformation materials: cutting off plant leaves in an ultra-clean bench by scissors, taking small discs with obvious main veins by using a sterile 6mm puncher, and putting the small discs into a normal culture medium for standby so as to prevent water dispersion;

3) Taking out the stored carrier agrobacterium bacterial liquid from a refrigerator with ultralow temperature of minus 80 ℃ in a laboratory, placing the carrier agrobacterium bacterial liquid on ice for melting, using a sterilized inoculating needle, taking a small amount of bacterial liquid, dividing the bacterial liquid on a prepared YEB solid culture medium with the concentration of 100mg/L Kan and 50mg/LRif, sealing the solid culture medium, inversely placing the solid culture medium in a 28 ℃ incubator for culturing for 48 hours, picking single bacterial colonies after single bacterial colonies grow out, shaking the bacterial liquid in LB liquid culture medium (200 mL) containing 100mg/L Kan and 50mg/L Rif overnight (a shaking table with the temperature of 28 ℃), and keeping the bacterial liquid to expand in logarithmic phase;

4) Pouring the bacterial liquid into a sterilized centrifugal bottle, centrifuging for 30min (2560 rpm), discarding the upper bacterial liquid, and suspending the precipitate (OD 600 = 0.3-0.4) with the prepared suspension;

5) Culturing the suspended bacterial liquid in a shaking table with the temperature of 50-1000rmp for 1h;

6) The explant or tissue culture material (with or without 2-7d preculture) is soaked in the suspended bacterial liquid for dip dyeing: shaking in a 50rmp shaking table for 1-2h at room temperature;

7) After the dip dyeing is finished, sucking redundant dip dyeing liquid into the leaf by using sterile filter paper, transferring the leaf into a differentiation culture medium, and culturing in a dark inversion mode for 2d, wherein the optimal temperature is 19-21 ℃;

8) After two days, the agrobacterium grows out of the periphery of the dip-dyed material, the material is required to be cleaned, the material is cleaned by sterile water for four times, the filtered washing liquid is used for cleaning once, and after the washing liquid is added, the material is placed in a shaking table with 50-1000rmp for cleaning for 1h;

9) Placing the cleaned material in a differentiation medium containing 500 mg/Lcefotam and 2mg/L Hyg of antibiotics, culturing in dark for 2 weeks, and culturing under light for two weeks;

10 After callus growth, transferring the material to an elongation culture medium containing 50mg/L Hyg, and replacing the culture medium every 2-4 weeks;

11 When the height of the material is 2cm, transferring the material into a rooting culture medium containing 2mg/L Hyg, wherein the whole transformation process is 3-8 months;

acquisition and statistics of transgenic plants

Respectively obtaining 40 PagPDS-2x35S-Cas9 and PagPDS-MAS-Cas9 transgenic plants by an agrobacterium transformation leaf disc method, wherein the transgenic plants of the two vectors have albino phenotype; as shown in fig. 6, which is a phenotype diagram of PagPDS-2x35s/MAS-Cas9 mediated transgenic plants in example 1 of the present invention, scale bar = 1 cm;

table 3 is the phenotype statistics of PagPDS-2x35S/MAS-Cas9 mediated transgenic plants, wherein among the 40 transgenic plants in PagPDS-2x35S-Cas9, albino seedlings 27, mutation rate 67.5%, wherein 21 (77.8%) are pure-whitened phenotype and 6 (22.3%) are yellowish-green phenotype.

Of the 40 transgenic plants in PagPDS-MAS-Cas9, albino seedlings were 30, of which 23 (76.7%) were pure white-standard and 7 (23.3%) were yellow-green phenotypes.

TABLE 3PagPDS-2x 35S/MAS-Cas9 mediated phenotype statistics of transgenic plants

Step (5): directional editing of poplar genes by CRISPR system

1) Identification of transgenic plant DNA level

After the leaves are subjected to agrobacteria dip-dyeing, screening and growing in a screening culture medium containing Hyg, and identifying rooting resistant seedlings after the material is subjected to differentiation, elongation and rooting processes;

taking a small number of leaves of a rooting plant screened by Hyg, extracting DNA (deoxyribonucleic acid) for PCR (polymerase chain reaction) detection, taking a PCR amplification product with plasmid as a template as a positive control, taking a PCR amplification reaction product with unconverted wild plant gene DNA as a template as a negative control, taking a PCR reaction system as 20 mu L, uniformly mixing various components, and carrying out PCR reaction, wherein the PCR reaction procedure is as follows: after pre-denaturation at 98 ℃ for 5min, 30 cycles are carried out, wherein each cycle is denaturation at 98 ℃ for 5s, annealing at 62 ℃ for 5s and extension at 72 ℃ for 20s, finally extension at 72 ℃ is carried out for 1min, PCR products are detected by 1.0% agarose gel electrophoresis, and the observation results show that PCR detection primers are shown as sequences 5 and 6 in a sequence table; the destination band 396bp;

2) Mutation identification of transgenic plants

After a positive transgenic plant is obtained, specific primers, S_PDS_F and S_PDS_R, of which the sequences are respectively shown as sequences 7 and 8 in a sequence table are utilized, PCR amplification is carried out to obtain a DNA sequence fragment with potential target site modification, then the fragment is connected to a T carrier after electrophoresis separation, escherichia coli is transformed to obtain a single colony, plasmids are extracted for sequencing of the target site, and the sequencing result is compared with a control sequence to analyze mutation events; FIG. 7 shows the comparison of the sequencing result of the target site in example 1 of the present invention with the control sequence;

sanger sequencing was performed on the transgenic plant extract DNA, pagPDS-2x35S-Cas9 at the T1 site, and 53.75% of the mutations in all albino phenotype plants were single nucleotide insertions, and the rate of detection of at least one allelic mutation was 65% in 26 edited plants; 21 strains with simultaneous mutations of two alleles, including 20 homozygous mutations and 1 biallelic mutation; in the T2/T3 locus, mutation rate was 67.5%, including 21 bi-allelic mutations and 4 homozygous mutations, and a deletion of 2 to 5nt was found near PAM (FIG. 7A);

in the PagPDS-MAS-Cas9 system, 30 plants were albino phenotype, mutation rate was 75%, which is higher than editing efficiency of 2X35S-Cas9, pure-whitened plants 23 plants (76.7%), yellow-green phenotype plants 7 plants (23.3%) (Table 3); albino phenotype plants had alleles with significant large fragment deletions at T1 and T2/T3; regarding analysis of T1, mutations of at least one allele were detected in 29 plants (72.5%), with 22 mutations in both alleles, including 19 homozygous mutations and 3 bi-allelic mutations, with a mutation rate of 72.5% at the T2/T3 locus, including 20 homozygous mutations and 2 bi-allelic mutations (fig. 7B);

as shown in table 4, the mutation types for the target sites are summarized.

Table 4: summarizing mutation types at target sites

The beneficial effects are that:

unlike CRISPR/Cas9 editing systems of Golden Gate in the application study of pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing, the inventors used only two simple vectors that were successfully used to target multiple sites of PagPDS gene in 84K poplar, caMV 35S promoter driven pC1300-Cas9 system mutation rate was 67.5%, pC1300-MAS-Cas9 (backbone vector pC1300-MAS-Cas9, final vector pC1300-MAS-Cas 9-PagPDS) system mutation rate was 75%, higher than that reported by Fan et al (2015) in woody species, pC1300-MAS-Cas9 system showed higher efficiency compared to pC1300-2X35S-Cas9 (backbone vector pC1300-2X 35S-9, final vector pC1300-2X 35S-9-PagPDS) system, and especially Cas system with abundant editing events of Cas 1300-DNA was shown to be deleted in the large Cas system; the CRISPR/Cas9 system constructed by homotail zymography can directly, conveniently and efficiently generate plants with a plurality of target mutations; thus, it is envisioned that the use of the pC1300-MAS-Cas9 system will help to study the relationship between multiple genes, and that efficient gene editing tools developed by the present inventors will facilitate gene function studies of woody tree species.

Sequence listing

<110> university of Zhejiang

<120> application of pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1716

<212> DNA

<213> Artificial sequence (PagPDS form P. Glandulosa sequence)

<400> 1

gtttgcaggg ctgttgttac agttgttata gctgttgaat ttggttttgg agaaatgagt 60

gcattgaact tgagctggca tagtaaatca ttagactctc aagttgcctt gagatgtggc 120

gcttatccta cttgttctca ccaaacgaat gcactagctt ttagaggcag tgaatcaatg 180

ggccattctt tgaaattccc atttggaaat tcttctgcta aaacaagact aaggaatcat 240

atccgccctc ctttgcgggt gcgtagttct ctacactaca gggtaattat tagttgccaa 300

tcaatacgtg aaaatttggg gtgatctttt gtctacgctg taggttgtct gtatggacta 360

tccaagaccg gaccttgata acacggtgaa tttcttagag gctgccttgt tatcttcatc 420

ctttcgttct tctccgcgtc cagctaaacc attaaatgtt gtcattgctg gtgcaggtga 480

tgaaatctta tccttttttt gtatgggaaa aaactgtgtt gattatttag attgatttct 540

atctatctgt aaaacttttt cgttgaaata ttgtcctcgt gtttaattaa taattttgaa 600

gctcgtactt tcacaagatt tttgcatgta ctcttgagag attgtgcgat acttacttgt 660

gtgtgactac aaattttcct tattttttca ggtttggcgg gtttatcgac tgcaaaatac 720

ttggcagatg cgggccataa gcctatattg cttgaagcaa gagatgtttt aggtggaaag 780

gtttttacac tgctctactt ttacccactt ctcaagttga caacatactt gtgcttgctt 840

atttttcaat tcatttgtta tttctcttag gttgtttttt gtcctatcat catgtcatta 900

caatataatt tataatttct tgttgtgctt gatgatgtaa atgttaataa tctgaaggga 960

tattgtgttc ttatatttgt ctaaatggtg tgcaataaat cttaataatg aaggaggtta 1020

ctgttttcct cactctttcc ttgggatgaa atgacgcaag gatctcatct tctgctttgt 1080

tcatcagatc tatctttttt gttattttat caactgatat ttggcctgtt ttctgatagg 1140

tggctgcatg gaaagatgat gatggagact ggtacgagac aggcttgcat atattctgta 1200

agttctcatt actagtaaaa ggaaatcggg gatagaattt taagcttggt tttttttttc 1260

ctctcacttc gatatgcctg ttcattaggt aagataggtt caaattcctt tgtgaattct 1320

gctgcatctt cactttataa tgccttttca aattcatcct tactttattt gctctttaat 1380

gtgtttttgt ggaaagtgct aaacataccc ctgctttatg gtttttctgt tcccattctg 1440

gctattgtaa tctagctgtt gatttttatg cttcatgaag ttgtttcttt cttttttagg 1500

tgtttacctt tggtgatttt atcgttgatc ttattgaatc tttgttttgt ttcttgcaag 1560

ttggggcata tccaaatgtg cagaatcttt ttggtgaact tggtatcaat gataggttgc 1620

aatggaagga gcattctatg atatttgcaa tgccaaataa gccaggagaa ttcagtcgat 1680

ttgattttcc tgaagttctc cctgcaccat taaatg 1716

<210> 2

<211> 1717

<212> DNA

<213> Artificial sequence (PagPDS form P. Alba sequence)

<400> 2

gtttgcaggg ctgttgttac agttgttata gctgttgaat ttggttttgg agaaatgagt 60

gcattgaact tgagctggca tagtaaatca ttagactctc aagttgcctt gagatgtggc 120

gcttatccta cttgttctca ccaaacgaat gcactagctt ttagaggcag tgaatcaatg 180

ggccattctt tgaaattccc atttggaaat tcttctgcta aaacaagact aaggaatcat 240

atccgccctc ctttgcgggt gcgtagttct ctacactaca gggtaattat tagttgccaa 300

tcaatacgtg aaaatttggg gtgatctttt gtctacgctg taggttgtct gtatggacta 360

tccaagaccg gaccttgata acacggtgaa tttcttagag gctgccttgt tatcttcatc 420

ctttcgttct tctccgcgtc cagctaaacc attaaatgtt gtcattgctg gtgcaggtga 480

tgaaatctta tccttttttt gtatgggaaa aaactgtgtt gattatttag attgatttct 540

atctatctgt aaaacttttt cgttgaaata ttgtcctcgt gtttaattaa taattttgaa 600

gctcgtactt tcacaagatt tttgcatgta ctcttgagag attgtgcgat acttacttgt 660

gtgtgactaa aaattttcct tattttttca ggtttggcgg gtttatcgac tgcaaaatac 720

ttggcagatg cgggccataa gcctatattg cttgaagcaa gagatgtttt aggtggaaag 780

gtttttacac tgctctactt ttacccactt ctcaagttga caacatactt gtgcttgctt 840

atttttcaat tcatttgtta tttctcttag gttgtttttt gtcctatcat cattgtcatt 900

acaatataat ttataatttc ttgttgtgct tgatgatgta aatgttaata atctgaaggg 960

atattgtgtt cttatatttg tctaaatggt gtgcaataaa tcttaataat gaaggaggtt 1020

actgttttcc tcactctttc cttgggatga aatgacgcaa ggatctcatc ttctgctttg 1080

ttcatcagat ctatcttttt tgttatttta tcaactgata tttggcctgt tttctgatag 1140

gtggctgcat ggaaagatga tgatggagac tggtacgaga caggcttgca tatattctgt 1200

aagttctcat tactagtaaa aggaaatcgg ggatagaatt ttaagcttgg tatttttttt 1260

cctctcactt cgatatgcct gttcattagg taagataggt tcaaattcct ttgtgaattc 1320

tgctgcatct tcactttata atgccttttc aaattcatcc ttactttatt tgctctttaa 1380

tgtgtttttg tggaaagtgc taaacatacc cctgctttat ggtttttctg ttcccattct 1440

ggctattgta atctagctgt tgatttttat gcttcatgaa gttgtttctt tcttttttag 1500

gtgtttacct ttggtgattt tatcgttgat cttattgaat ctttgttttg tttcttgcaa 1560

gttggggcat atccaaatgt gcagaatctt tttggtgaac ttggtatcaa tgataggttg 1620

caatggaagg agcattctat gatatttgca atgccaaata agccaggaga attcagtcga 1680

tttgattttc ctgaagttct ccctgcacca ttaaatg 1717

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (PagPDS_F)

<400> 3

aactgggtat gcgaagactt cc 22

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (PagPDS_R)

<400> 4

aagcaagcac aagtatgttg tcaac 25

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (Cas 9F)

<400> 5

gaaaggtcgg tattcacggc gtt 23

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence (Cas 9R)

<400> 6

tgttgccaaa gataggatgc ct 22

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (S_PDS_F)

<400> 7

gattatgcgt ttcgtttctt ggct 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (S_PDS_R)

<400> 8

gatttcatca cctgcaccag caat 24

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence (gRNA 1)

<400> 9

ctatgccagc tcaagttcaa tgca 24

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence (gRNA 2)

<400> 10

gaggcagtga atcaatgggc catt 24

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (gRNA 3)

<400> 11

gttcttctcc gcgtccagct aaac 24

<210> 12

<211> 3502

<212> DNA

<213> Artificial sequence (SK-gRNA)

<400> 12

ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60

ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 180

ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 240

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 300

gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 360

tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 420

ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660

actagtggat ccctcgagaa gcttcgttga acaacggaaa ctcgacttgc cttccgcaca 720

atacatcatt tcttcttagc tttttttctt cttcttcgtt catacagttt ttttttgttt 780

atcagcttac attttcttga accgtagctt tcgttttctt ctttttaact ttccattcgg 840

agtttttgta tcttgtttca tagtttgtcc caggattaga atgattaggc atcgaacctt 900

caagaatttg attgaataaa acatcttcat tcttaagata tgaagataat cttcaaaagg 960

cccctgggaa tctgaaagaa gagaagcagg cccatttata tgggaaagaa caatagtatt 1020

tcttatatag gcccatttaa gttgaaaaca atcttcaaaa gtcccacatc gcttagataa 1080

gaaaacgaag ctgagtttat atacagctag agtcgaagta gtgattggct cgcaggtgaa 1140

cacaacacct gcacacgttt cagagctatg ctggaaacag catagcaagt tgaaataagg 1200

ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc cacataatcg 1260

tcgacagatc ttctagagcg gccgccaccg cggtggagct ccagcttttg ttccctttag 1320

tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 1380

tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 1440

gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 1500

ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 1560

cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 1620

cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 1680

aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1740

gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1800

tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1860

agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1920

ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1980

taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 2040

gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 2100

gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 2160

ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 2220

ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2280

gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2340

caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 2400

taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 2460

aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 2520

tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 2580

tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 2640

gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 2700

gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 2760

aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 2820

gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 2880

ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 2940

tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 3000

atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 3060

ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 3120

ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 3180

ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 3240

atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 3300

gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3360

tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 3420

ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 3480

acatttcccc gaaaagtgcc ac 3502

<210> 13

<211> 16078

<212> DNA

<213> Artificial sequence (pC 1300-MAS-Cas 9-PagPDS)

<400> 13

aattcgagct cggtaccgga ctagtggatc cctcgagaag cttcgttgaa caacggaaac 60

tcgacttgcc ttccgcacaa tacatcattt cttcttagct ttttttcttc ttcttcgttc 120

atacagtttt tttttgttta tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc 180

tttttaactt tccattcgga gtttttgtat cttgtttcat agtttgtccc aggattagaa 240

tgattaggca tcgaaccttc aagaatttga ttgaataaaa catcttcatt cttaagatat 300

gaagataatc ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat 360

gggaaagaac aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag 420

tcccacatcg cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag 480

tgattggagt gcattgaact tgagcgtttc agagctatgc tggaaacagc atagcaagtt 540

gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc 600

acataatcgt cgagaagctt cgttgaacaa cggaaactcg acttgccttc cgcacaatac 660

atcatttctt cttagctttt tttcttcttc ttcgttcata cagttttttt ttgtttatca 720

gcttacattt tcttgaaccg tagctttcgt tttcttcttt ttaactttcc attcggagtt 780

tttgtatctt gtttcatagt ttgtcccagg attagaatga ttaggcatcg aaccttcaag 840

aatttgattg aataaaacat cttcattctt aagatatgaa gataatcttc aaaaggcccc 900

tgggaatctg aaagaagaga agcaggccca tttatatggg aaagaacaat agtatttctt 960

atataggccc atttaagttg aaaacaatct tcaaaagtcc cacatcgctt agataagaaa 1020

acgaagctga gtttatatac agctagagtc gaagtagtga ttgtgttatc aaggtccggt 1080

ctgtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag tccgttatca 1140

acttgaaaaa gtggcaccga gtcggtgctt tttttccaca taatcgtcga cagatcttct 1200

agtggatccc tcgagaagct tcgttgaaca acggaaactc gacttgcctt ccgcacaata 1260

catcatttct tcttagcttt ttttcttctt cttcgttcat acagtttttt tttgtttatc 1320

agcttacatt ttcttgaacc gtagctttcg ttttcttctt tttaactttc cattcggagt 1380

ttttgtatct tgtttcatag tttgtcccag gattagaatg attaggcatc gaaccttcaa 1440

gaatttgatt gaataaaaca tcttcattct taagatatga agataatctt caaaaggccc 1500

ctgggaatct gaaagaagag aagcaggccc atttatatgg gaaagaacaa tagtatttct 1560

tatataggcc catttaagtt gaaaacaatc ttcaaaagtc ccacatcgct tagataagaa 1620

aacgaagctg agtttatata cagctagagt cgaagtagtg attgtcaccg tgttatcaag 1680

gtcgtttcag agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc 1740

aacttgaaaa agtggcaccg agtcggtgct ttttttccac ataatcgtcg acagatccgt 1800

acctgatctc tttctcgatc gagggagatt tttcaaatca gtgcgcaaga cgtgacgtaa 1860

gtatccgagt cagtttttat ttttctacta atttggtcgt ttatttcggc gtgtaggaca 1920

tggcaaccgg gcctgaattt cgcgggtatt ctgtttctat tccaactttt tcttgatccg 1980

cagccattaa cgacttttga atagatacgc tgacacgcca agcctcgcta gtcaaaagtg 2040

taccaaacaa cgctttacag caagaacgga atgcgcgtga cgctcgcggt gacgccattt 2100

cgccttttca gaaatggata aatagccttg cttcctatta tatcttccca aattaccaat 2160

acattacact agcatctgaa tttcataacc aatctcgata caccaaatcg accaagctag 2220

atccatggcc cctaagaaga agagaaaggt cggtattcac ggcgttcctg cggcgatgga 2280

caagaagtat agtattggtc tggacattgg gacgaattcc gttggctggg ccgtgatcac 2340

cgatgagtac aaggtccctt ccaagaagtt taaggttctg gggaacaccg atcggcacag 2400

catcaagaag aatctcattg gagccctcct gttcgactca ggcgagaccg ccgaagcaac 2460

aaggctcaag agaaccgcaa ggagacggta tacaagaagg aagaatagga tctgctacct 2520

gcaggagatt ttcagcaacg aaatggcgaa ggtggacgat tcgttctttc atagattgga 2580

ggagagtttc ctcgtcgagg aagataagaa gcacgagagg catcctatct ttggcaacat 2640

tgtcgacgag gttgcctatc acgaaaagta ccccacaatc tatcatctgc ggaagaagct 2700

tgtggactcg actgataagg cggaccttag attgatctac ctcgctctgg cacacatgat 2760

taagttcagg ggccattttc tgatcgaggg ggatcttaac ccggacaata gcgatgtgga 2820

caagttgttc atccagctcg tccaaaccta caatcagctc tttgaggaaa acccaattaa 2880

tgcttcaggc gtcgacgcca aggcgatcct gtctgcacgc ctttcaaagt ctcgccggct 2940

tgagaacttg atcgctcaac tcccgggcga aaagaagaac ggcttgttcg ggaatctcat 3000

tgcactttcg ttggggctca caccaaactt caagagtaat tttgatctcg ctgaggacgc 3060

aaagctgcag ctttccaagg acacttatga cgatgacctg gataaccttt tggcccaaat 3120

cggcgatcag tacgcggact tgttcctcgc cgcgaagaat ttgtcggacg cgatcctcct 3180

gagtgatatt ctccgcgtga acaccgagat tacaaaggcc ccgctctcgg cgagtatgat 3240

caagcgctat gacgagcacc atcaggatct gacccttttg aaggctttgg tccggcagca 3300

actcccagag aagtacaagg aaatcttctt tgatcaatcc aagaacggct acgctggtta 3360

tattgacggc ggggcatcgc aggaggaatt ctacaagttt atcaagccaa ttctggagaa 3420

gatggatggc acagaggaac tcctggtgaa gctcaatagg gaggaccttt tgcggaagca 3480

aagaactttc gataacggca gcatccctca ccagattcat ctcggggagc tgcacgccat 3540

cctgagaagg caggaagact tctacccctt tcttaaggat aaccgggaga agatcgaaaa 3600

gattctgacg ttcagaattc cgtactatgt cggaccactc gcccggggta attccagatt 3660

tgcgtggatg accagaaaga gcgaggaaac catcacacct tggaacttcg aggaagtggt 3720

cgataagggc gcttccgcac agagcttcat tgagcgcatg acaaattttg acaagaacct 3780

gcctaatgag aaggtccttc ccaagcattc cctcctgtac gagtatttca ctgtttataa 3840

cgaactcacg aaggtgaagt atgtgaccga gggaatgcgc aagcccgcct tcctgagcgg 3900

cgagcaaaag aaggcgatcg tggacctttt gtttaagacc aatcggaagg tcacagttaa 3960

gcagctcaag gaggactact tcaagaagat tgaatgcttc gattccgttg agatcagcgg 4020

cgtggaagac aggtttaacg cgtcactggg gacttaccac gatctcctga agatcattaa 4080

ggataaggac ttcttggaca acgaggaaaa tgaggatatc ctcgaagaca ttgtcctgac 4140

tcttacgttg tttgaggata gggaaatgat cgaggaacgc ttgaagacgt atgcccatct 4200

cttcgatgac aaggttatga agcagctcaa gagaagaaga tacaccggat ggggaaggct 4260

gtcccgcaag cttatcaatg gcattagaga caagcaatca gggaagacaa tccttgactt 4320

tttgaagtct gatggcttcg cgaacaggaa ttttatgcag ctgattcacg atgactcact 4380

tactttcaag gaggatatcc agaaggctca agtgtcggga caaggtgaca gtctgcacga 4440

gcatatcgcc aaccttgcgg gatctcctgc aatcaagaag ggtattctgc agacagtcaa 4500

ggttgtggat gagcttgtga aggtcatggg acggcataag cccgagaaca tcgttattga 4560

gatggccaga gaaaatcaga ccacacaaaa gggtcagaag aactcgaggg agcgcatgaa 4620

gcgcatcgag gaaggcatta aggagctggg gagtcagatc cttaaggagc acccggtgga 4680

aaacacgcag ttgcaaaatg agaagctcta tctgtactat ctgcaaaatg gcagggatat 4740

gtatgtggac caggagttgg atattaaccg cctctcggat tacgacgtcg atcatatcgt 4800

tcctcagtcc ttccttaagg atgacagcat tgacaataag gttctcacca ggtccgacaa 4860

gaaccgcggg aagtccgata atgtgcccag cgaggaagtc gttaagaaga tgaagaacta 4920

ctggaggcaa cttttgaatg ccaagttgat cacacagagg aagtttgata acctcactaa 4980

ggccgagcgc ggaggtctca gcgaactgga caaggcgggc ttcattaagc ggcaactggt 5040

tgagactaga cagatcacga agcacgtggc gcagattctc gattcacgca tgaacacgaa 5100

gtacgatgag aatgacaagc tgatccggga agtgaaggtc atcaccttga agtcaaagct 5160

cgtttctgac ttcaggaagg atttccaatt ttataaggtg cgcgagatca acaattatca 5220

ccatgctcat gacgcatacc tcaacgctgt ggtcggaaca gcattgatta agaagtaccc 5280

gaagctcgag tccgaattcg tgtacggtga ctataaggtt tacgatgtgc gcaagatgat 5340

cgccaagtca gagcaggaaa ttggcaaggc cactgcgaag tatttctttt actctaacat 5400

tatgaatttc tttaagactg agatcacgct ggctaatggc gaaatccgga agagaccact 5460

tattgagacc aacggcgaga caggggaaat cgtgtgggac aaggggaggg atttcgccac 5520

agtccgcaag gttctctcta tgcctcaagt gaatattgtc aagaagactg aagtccagac 5580

gggcgggttc tcaaaggaat ctattctgcc caagcggaac tcggataagc ttatcgccag 5640

aaagaaggac tgggacccga agaagtatgg aggtttcgac tcaccaacgg tggcttactc 5700

tgtcctggtt gtggcaaagg tggagaaggg aaagtcaaag aagctcaagt ctgtcaagga 5760

gctcctgggt atcaccatta tggagaggtc cagcttcgaa aagaatccga tcgattttct 5820

cgaggcgaag ggatataagg aagtgaagaa ggacctgatc attaagcttc caaagtacag 5880

tcttttcgag ttggaaaacg gcaggaagcg catgttggct tccgcaggag agctccagaa 5940

gggtaacgag cttgctttgc cgtccaagta tgtgaacttc ctctatctgg catcccacta 6000

cgagaagctc aagggcagcc cagaggataa cgaacagaag caactgtttg tggagcaaca 6060

caagcattat cttgacgaga tcattgaaca gatttcggag ttcagtaagc gcgtcatcct 6120

cgccgacgcg aatttggata aggttctctc agcctacaac aagcaccggg acaagcctat 6180

cagagagcag gcggaaaata tcattcatct cttcaccctg acaaaccttg gggctcccgc 6240

tgcattcaag tattttgaca ctacgattga tcggaagaga tacacttcta cgaaggaggt 6300

gctggatgca acccttatcc accaatcgat tactggcctc tacgagacgc ggatcgactt 6360

gagtcagctc gggggggata agagaccagc ggcaaccaag aaggcaggac aagcgaagaa 6420

gaagaagtag caattcggta cgctgaaatc accagtctct ctctacaaat ctatctctct 6480

ctattttctc cataaataat gtgtgagtag tttcccgata agggaaatta gggttcttat 6540

agggtttcgc tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa 6600

atacttctat caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat 6660

ctcctaaagt ccctatagat ctttgtcgtg aatataaacc agacacgaga cgactaaacc 6720

tggagcccag acgccgttcg aagctagaag taccgcttag gcaggaggcc gttagggaaa 6780

agatgctaag gcagggttgg ttacgttgac tcccccgtag gtttggttta aatatgatga 6840

agtggacgga aggaaggagg aagacaagga aggataaggt tgcaggccct gtgcaaggta 6900

agaagatgga aatttgatag aggtacgcta ctatacttat actatacgct aagggaatgc 6960

ttgtatttat accctatacc ccctaataac cccttatcaa tttaagaaat aatccgcata 7020

agcccccgct taaaaattgg tatcagagcc atgaataggt ctatgaccaa aactcaagag 7080

gataaaacct caccaaaata cgaaagagtt cttaactcta aagataaaag atctttcaag 7140

atcaaaacta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg ttttacaacg 7200

tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 7260

cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 7320

cctgaatggc gaatgctaga gcagcttgag cttggatcag attgtcgttt cccgccttca 7380

gtttaaacta tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt 7440

ttattagaat aacggatatt taaaagggcg tgaaaaggtt tatccgttcg tccatttgta 7500

tgtgcatgcc aaccacaggg ttcccctcgg gatcaaagta ctttgatcca acccctccgc 7560

tgctatagtg cagtcggctt ctgacgttca gtgcagccgt cttctgaaaa cgacatgtcg 7620

cacaagtcct aagttacgcg acaggctgcc gccctgccct tttcctggcg ttttcttgtc 7680

gcgtgtttta gtcgcataaa gtagaatact tgcgactaga accggagaca ttacgccatg 7740

aacaagagcg ccgccgctgg cctgctgggc tatgcccgcg tcagcaccga cgaccaggac 7800

ttgaccaacc aacgggccga actgcacgcg gccggctgca ccaagctgtt ttccgagaag 7860

atcaccggca ccaggcgcga ccgcccggag ctggccagga tgcttgacca cctacgccct 7920

ggcgacgttg tgacagtgac caggctagac cgcctggccc gcagcacccg cgacctactg 7980

gacattgccg agcgcatcca ggaggccggc gcgggcctgc gtagcctggc agagccgtgg 8040

gccgacacca ccacgccggc cggccgcatg gtgttgaccg tgttcgccgg cattgccgag 8100

ttcgagcgtt ccctaatcat cgaccgcacc cggagcgggc gcgaggccgc caaggcccga 8160

ggcgtgaagt ttggcccccg ccctaccctc accccggcac agatcgcgca cgcccgcgag 8220

ctgatcgacc aggaaggccg caccgtgaaa gaggcggctg cactgcttgg cgtgcatcgc 8280

tcgaccctgt accgcgcact tgagcgcagc gaggaagtga cgcccaccga ggccaggcgg 8340

cgcggtgcct tccgtgagga cgcattgacc gaggccgacg ccctggcggc cgccgagaat 8400

gaacgccaag aggaacaagc atgaaaccgc accaggacgg ccaggacgaa ccgtttttca 8460

ttaccgaaga gatcgaggcg gagatgatcg cggccgggta cgtgttcgag ccgcccgcgc 8520

acgtctcaac cgtgcggctg catgaaatcc tggccggttt gtctgatgcc aagctggcgg 8580

cctggccggc cagcttggcc gctgaagaaa ccgagcgccg ccgtctaaaa aggtgatgtg 8640

tatttgagta aaacagcttg cgtcatgcgg tcgctgcgta tatgatgcga tgagtaaata 8700

aacaaatacg caaggggaac gcatgaaggt tatcgctgta cttaaccaga aaggcgggtc 8760

aggcaagacg accatcgcaa cccatctagc ccgcgccctg caactcgccg gggccgatgt 8820

tctgttagtc gattccgatc cccagggcag tgcccgcgat tgggcggccg tgcgggaaga 8880

tcaaccgcta accgttgtcg gcatcgaccg cccgacgatt gaccgcgacg tgaaggccat 8940

cggccggcgc gacttcgtag tgatcgacgg agcgccccag gcggcggact tggctgtgtc 9000

cgcgatcaag gcagccgact tcgtgctgat tccggtgcag ccaagccctt acgacatatg 9060

ggccaccgcc gacctggtgg agctggttaa gcagcgcatt gaggtcacgg atggaaggct 9120

acaagcggcc tttgtcgtgt cgcgggcgat caaaggcacg cgcatcggcg gtgaggttgc 9180

cgaggcgctg gccgggtacg agctgcccat tcttgagtcc cgtatcacgc agcgcgtgag 9240

ctacccaggc actgccgccg ccggcacaac cgttcttgaa tcagaacccg agggcgacgc 9300

tgcccgcgag gtccaggcgc tggccgctga aattaaatca aaactcattt gagttaatga 9360

ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg ccggccgtcc gagcgcacgc 9420

agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc cagccatgaa gcgggtcaac 9480

tttcagttgc cggcggagga tcacaccaag ctgaagatgt acgcggtacg ccaaggcaag 9540

accattaccg agctgctatc tgaatacatc gcgcagctac cagagtaaat gagcaaatga 9600

ataaatgagt agatgaattt tagcggctaa aggaggcggc atggaaaatc aagaacaacc 9660

aggcaccgac gccgtggaat gccccatgtg tggaggaacg ggcggttggc caggcgtaag 9720

cggctgggtt gtctgccggc cctgcaatgg cactggaacc cccaagcccg aggaatcggc 9780

gtgacggtcg caaaccatcc ggcccggtac aaatcggcgc ggcgctgggt gatgacctgg 9840

tggagaagtt gaaggccgcg caggccgccc agcggcaacg catcgaggca gaagcacgcc 9900

ccggtgaatc gtggcaagcg gccgctgatc gaatccgcaa agaatcccgg caaccgccgg 9960

cagccggtgc gccgtcgatt aggaagccgc ccaagggcga cgagcaacca gattttttcg 10020

ttccgatgct ctatgacgtg ggcacccgcg atagtcgcag catcatggac gtggccgttt 10080

tccgtctgtc gaagcgtgac cgacgagctg gcgaggtgat ccgctacgag cttccagacg 10140

ggcacgtaga ggtttccgca gggccggccg gcatggccag tgtgtgggat tacgacctgg 10200

tactgatggc ggtttcccat ctaaccgaat ccatgaaccg ataccgggaa gggaagggag 10260

acaagcccgg ccgcgtgttc cgtccacacg ttgcggacgt actcaagttc tgccggcgag 10320

ccgatggcgg aaagcagaaa gacgacctgg tagaaacctg cattcggtta aacaccacgc 10380

acgttgccat gcagcgtacg aagaaggcca agaacggccg cctggtgacg gtatccgagg 10440

gtgaagcctt gattagccgc tacaagatcg taaagagcga aaccgggcgg ccggagtaca 10500

tcgagatcga gctagctgat tggatgtacc gcgagatcac agaaggcaag aacccggacg 10560

tgctgacggt tcaccccgat tactttttga tcgatcccgg catcggccgt tttctctacc 10620

gcctggcacg ccgcgccgca ggcaaggcag aagccagatg gttgttcaag acgatctacg 10680

aacgcagtgg cagcgccgga gagttcaaga agttctgttt caccgtgcgc aagctgatcg 10740

ggtcaaatga cctgccggag tacgatttga aggaggaggc ggggcaggct ggcccgatcc 10800

tagtcatgcg ctaccgcaac ctgatcgagg gcgaagcatc cgccggttcc taatgtacgg 10860

agcagatgct agggcaaatt gccctagcag gggaaaaagg tcgaaaaggt ctctttcctg 10920

tggatagcac gtacattggg aacccaaagc cgtacattgg gaaccggaac ccgtacattg 10980

ggaacccaaa gccgtacatt gggaaccggt cacacatgta agtgactgat ataaaagaga 11040

aaaaaggcga tttttccgcc taaaactctt taaaacttat taaaactctt aaaacccgcc 11100

tggcctgtgc ataactgtct ggccagcgca cagccgaaga gctgcaaaaa gcgcctaccc 11160

ttcggtcgct gcgctcccta cgccccgccg cttcgcgtcg gcctatcgcg gccgctggcc 11220

gctcaaaaat ggctggccta cggccaggca atctaccagg gcgcggacaa gccgcgccgt 11280

cgccactcga ccgccggcgc ccacatcaag gcaccctgcc tcgcgcgttt cggtgatgac 11340

ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat 11400

gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca 11460

gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag 11520

agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga 11580

gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 11640

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 11700

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 11760

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 11820

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 11880

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 11940

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 12000

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12060

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12120

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12180

cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 12240

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 12300

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 12360

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 12420

actcacgtta agggattttg gtcatgcatt ctaggtacta aaacaattca tccagtaaaa 12480

tataatattt tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga 12540

catactgttc ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc 12600

acttgtccgc cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca 12660

caaagatgtt gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt 12720

ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa tggagtgtct tcttcccagt 12780

tttcgcaatc cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc 12840

tgtctaagct attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc 12900

actccgcata cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc 12960

aaaggacgcc atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt 13020

gcaggacctt tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt 13080

ccacatcata ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat 13140

tttctcccac cagcttatat accttagcag gagacattcc ttccgtatct tttacgcagc 13200

ggtatttttc gatcagtttt ttcaattccg gtgatattct cattttagcc atttattatt 13260

tccttcctct tttctacagt atttaaagat accccaagaa gctaattata acaagacgaa 13320

ctccaattca ctgttccttg cattctaaaa ccttaaatac cagaaaacag ctttttcaaa 13380

gttgttttca aagttggcgt ataacatagt atcgacggag ccgattttga aaccgcggtg 13440

atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc gcgagatcat 13500

ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg taacatgagc 13560

aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga tgggctgcct 13620

gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc 13680

aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg 13740

tttttaatgt actgaattaa cgccgaatta attcggggga tctggatttt agtactggat 13800

tttggtttta ggaattagaa attttattga tagaagtatt ttacaaatac aaatacatac 13860

taagggtttc ttatatgctc aacacatgag cgaaacccta taggaaccct aattccctta 13920

tctgggaact actcacacat tattatggag aaactcgagc ttgtcgatcg acagatccgg 13980

tcggcatcta ctctatttct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc 14040

ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt 14100

acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc 14160

tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag 14220

catatacgcc cggagtcgtg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg 14280

cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta 14340

ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc 14400

cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc 14460

ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc 14520

catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca 14580

tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag 14640

atcggccgca gcgatcgcat ccatagcctc cgcgaccggt tgtagaacag cgggcagttc 14700

ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag 14760

gctctcgcta aactccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa 14820

gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac 14880

atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg 14940

catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt 15000

gagttcaggc tttttcatat ctcattgccc cccgggatct gcgaaagctc gagagagata 15060

gatttgtaga gagagactgg tgatttcagc gtgtcctctc caaatgaaat gaacttcctt 15120

atatagagga aggtcttgcg aaggatagtg ggattgtgcg tcatccctta cgtcagtgga 15180

gatatcacat caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat 15240

gctcctcgtg ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat 15300

agcctttcct ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct 15360

tttgatgaag tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct 15420

ttgttgaaaa gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga 15480

gtagacgaga gtgtcgtgct ccaccatgtt atcacatcaa tccacttgct ttgaagacgt 15540

ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc 15600

actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta 15660

ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa 15720

tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc 15780

tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttggc 15840

aagctgctct agccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 15900

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 15960

agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 16020

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacg 16078

<210> 14

<211> 16406

<212> DNA

<213> Artificial sequence (pC 1300-2X35S-Cas 9-PagPDS)

<400> 14

aattcgagct cggtaccgga ctagtggatc cctcgagaag cttcgttgaa caacggaaac 60

tcgacttgcc ttccgcacaa tacatcattt cttcttagct ttttttcttc ttcttcgttc 120

atacagtttt tttttgttta tcagcttaca ttttcttgaa ccgtagcttt cgttttcttc 180

tttttaactt tccattcgga gtttttgtat cttgtttcat agtttgtccc aggattagaa 240

tgattaggca tcgaaccttc aagaatttga ttgaataaaa catcttcatt cttaagatat 300

gaagataatc ttcaaaaggc ccctgggaat ctgaaagaag agaagcaggc ccatttatat 360

gggaaagaac aatagtattt cttatatagg cccatttaag ttgaaaacaa tcttcaaaag 420

tcccacatcg cttagataag aaaacgaagc tgagtttata tacagctaga gtcgaagtag 480

tgattggagt gcattgaact tgagcgtttc agagctatgc tggaaacagc atagcaagtt 540

gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcc 600

acataatcgt cgagaagctt cgttgaacaa cggaaactcg acttgccttc cgcacaatac 660

atcatttctt cttagctttt tttcttcttc ttcgttcata cagttttttt ttgtttatca 720

gcttacattt tcttgaaccg tagctttcgt tttcttcttt ttaactttcc attcggagtt 780

tttgtatctt gtttcatagt ttgtcccagg attagaatga ttaggcatcg aaccttcaag 840

aatttgattg aataaaacat cttcattctt aagatatgaa gataatcttc aaaaggcccc 900

tgggaatctg aaagaagaga agcaggccca tttatatggg aaagaacaat agtatttctt 960

atataggccc atttaagttg aaaacaatct tcaaaagtcc cacatcgctt agataagaaa 1020

acgaagctga gtttatatac agctagagtc gaagtagtga ttgtgttatc aaggtccggt 1080

ctgtttcaga gctatgctgg aaacagcata gcaagttgaa ataaggctag tccgttatca 1140

acttgaaaaa gtggcaccga gtcggtgctt tttttccaca taatcgtcga cagatcttct 1200

agtggatccc tcgagaagct tcgttgaaca acggaaactc gacttgcctt ccgcacaata 1260

catcatttct tcttagcttt ttttcttctt cttcgttcat acagtttttt tttgtttatc 1320

agcttacatt ttcttgaacc gtagctttcg ttttcttctt tttaactttc cattcggagt 1380

ttttgtatct tgtttcatag tttgtcccag gattagaatg attaggcatc gaaccttcaa 1440

gaatttgatt gaataaaaca tcttcattct taagatatga agataatctt caaaaggccc 1500

ctgggaatct gaaagaagag aagcaggccc atttatatgg gaaagaacaa tagtatttct 1560

tatataggcc catttaagtt gaaaacaatc ttcaaaagtc ccacatcgct tagataagaa 1620

aacgaagctg agtttatata cagctagagt cgaagtagtg attgtcaccg tgttatcaag 1680

gtcgtttcag agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc 1740

aacttgaaaa agtggcaccg agtcggtgct ttttttccac ataatcgtcg acagatccgt 1800

acccctactc caaaaatgtc aaagatacag tctcagaaga ccaaagggct attgagactt 1860

ttcaacaaag ggtaatttcg ggaaacctcc tcggattcca ttgcccagct atctgtcact 1920

tcatcgaaag gacagtagaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 1980

gaaaggctat cattcaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 2040

cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 2100

gtgacatctc cactgacgta agggatgacg cacaatccca cccctactcc aaaaatgtca 2160

aagatacagt ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatttcgg 2220

gaaacctcct cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa 2280

aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg 2340

cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 2400

aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa 2460

gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 2520

ttcatttgga gaggacagcc caagctagat ccatggcccc taagaagaag agaaaggtcg 2580

gtattcacgg cgttcctgcg gcgatggaca agaagtatag tattggtctg gacattggga 2640

cgaattccgt tggctgggcc gtgatcaccg atgagtacaa ggtcccttcc aagaagttta 2700

aggttctggg gaacaccgat cggcacagca tcaagaagaa tctcattgga gccctcctgt 2760

tcgactcagg cgagaccgcc gaagcaacaa ggctcaagag aaccgcaagg agacggtata 2820

caagaaggaa gaataggatc tgctacctgc aggagatttt cagcaacgaa atggcgaagg 2880

tggacgattc gttctttcat agattggagg agagtttcct cgtcgaggaa gataagaagc 2940

acgagaggca tcctatcttt ggcaacattg tcgacgaggt tgcctatcac gaaaagtacc 3000

ccacaatcta tcatctgcgg aagaagcttg tggactcgac tgataaggcg gaccttagat 3060

tgatctacct cgctctggca cacatgatta agttcagggg ccattttctg atcgaggggg 3120

atcttaaccc ggacaatagc gatgtggaca agttgttcat ccagctcgtc caaacctaca 3180

atcagctctt tgaggaaaac ccaattaatg cttcaggcgt cgacgccaag gcgatcctgt 3240

ctgcacgcct ttcaaagtct cgccggcttg agaacttgat cgctcaactc ccgggcgaaa 3300

agaagaacgg cttgttcggg aatctcattg cactttcgtt ggggctcaca ccaaacttca 3360

agagtaattt tgatctcgct gaggacgcaa agctgcagct ttccaaggac acttatgacg 3420

atgacctgga taaccttttg gcccaaatcg gcgatcagta cgcggacttg ttcctcgccg 3480

cgaagaattt gtcggacgcg atcctcctga gtgatattct ccgcgtgaac accgagatta 3540

caaaggcccc gctctcggcg agtatgatca agcgctatga cgagcaccat caggatctga 3600

cccttttgaa ggctttggtc cggcagcaac tcccagagaa gtacaaggaa atcttctttg 3660

atcaatccaa gaacggctac gctggttata ttgacggcgg ggcatcgcag gaggaattct 3720

acaagtttat caagccaatt ctggagaaga tggatggcac agaggaactc ctggtgaagc 3780

tcaataggga ggaccttttg cggaagcaaa gaactttcga taacggcagc atccctcacc 3840

agattcatct cggggagctg cacgccatcc tgagaaggca ggaagacttc tacccctttc 3900

ttaaggataa ccgggagaag atcgaaaaga ttctgacgtt cagaattccg tactatgtcg 3960

gaccactcgc ccggggtaat tccagatttg cgtggatgac cagaaagagc gaggaaacca 4020

tcacaccttg gaacttcgag gaagtggtcg ataagggcgc ttccgcacag agcttcattg 4080

agcgcatgac aaattttgac aagaacctgc ctaatgagaa ggtccttccc aagcattccc 4140

tcctgtacga gtatttcact gtttataacg aactcacgaa ggtgaagtat gtgaccgagg 4200

gaatgcgcaa gcccgccttc ctgagcggcg agcaaaagaa ggcgatcgtg gaccttttgt 4260

ttaagaccaa tcggaaggtc acagttaagc agctcaagga ggactacttc aagaagattg 4320

aatgcttcga ttccgttgag atcagcggcg tggaagacag gtttaacgcg tcactgggga 4380

cttaccacga tctcctgaag atcattaagg ataaggactt cttggacaac gaggaaaatg 4440

aggatatcct cgaagacatt gtcctgactc ttacgttgtt tgaggatagg gaaatgatcg 4500

aggaacgctt gaagacgtat gcccatctct tcgatgacaa ggttatgaag cagctcaaga 4560

gaagaagata caccggatgg ggaaggctgt cccgcaagct tatcaatggc attagagaca 4620

agcaatcagg gaagacaatc cttgactttt tgaagtctga tggcttcgcg aacaggaatt 4680

ttatgcagct gattcacgat gactcactta ctttcaagga ggatatccag aaggctcaag 4740

tgtcgggaca aggtgacagt ctgcacgagc atatcgccaa ccttgcggga tctcctgcaa 4800

tcaagaaggg tattctgcag acagtcaagg ttgtggatga gcttgtgaag gtcatgggac 4860

ggcataagcc cgagaacatc gttattgaga tggccagaga aaatcagacc acacaaaagg 4920

gtcagaagaa ctcgagggag cgcatgaagc gcatcgagga aggcattaag gagctgggga 4980

gtcagatcct taaggagcac ccggtggaaa acacgcagtt gcaaaatgag aagctctatc 5040

tgtactatct gcaaaatggc agggatatgt atgtggacca ggagttggat attaaccgcc 5100

tctcggatta cgacgtcgat catatcgttc ctcagtcctt ccttaaggat gacagcattg 5160

acaataaggt tctcaccagg tccgacaaga accgcgggaa gtccgataat gtgcccagcg 5220

aggaagtcgt taagaagatg aagaactact ggaggcaact tttgaatgcc aagttgatca 5280

cacagaggaa gtttgataac ctcactaagg ccgagcgcgg aggtctcagc gaactggaca 5340

aggcgggctt cattaagcgg caactggttg agactagaca gatcacgaag cacgtggcgc 5400

agattctcga ttcacgcatg aacacgaagt acgatgagaa tgacaagctg atccgggaag 5460

tgaaggtcat caccttgaag tcaaagctcg tttctgactt caggaaggat ttccaatttt 5520

ataaggtgcg cgagatcaac aattatcacc atgctcatga cgcatacctc aacgctgtgg 5580

tcggaacagc attgattaag aagtacccga agctcgagtc cgaattcgtg tacggtgact 5640

ataaggttta cgatgtgcgc aagatgatcg ccaagtcaga gcaggaaatt ggcaaggcca 5700

ctgcgaagta tttcttttac tctaacatta tgaatttctt taagactgag atcacgctgg 5760

ctaatggcga aatccggaag agaccactta ttgagaccaa cggcgagaca ggggaaatcg 5820

tgtgggacaa ggggagggat ttcgccacag tccgcaaggt tctctctatg cctcaagtga 5880

atattgtcaa gaagactgaa gtccagacgg gcgggttctc aaaggaatct attctgccca 5940

agcggaactc ggataagctt atcgccagaa agaaggactg ggacccgaag aagtatggag 6000

gtttcgactc accaacggtg gcttactctg tcctggttgt ggcaaaggtg gagaagggaa 6060

agtcaaagaa gctcaagtct gtcaaggagc tcctgggtat caccattatg gagaggtcca 6120

gcttcgaaaa gaatccgatc gattttctcg aggcgaaggg atataaggaa gtgaagaagg 6180

acctgatcat taagcttcca aagtacagtc ttttcgagtt ggaaaacggc aggaagcgca 6240

tgttggcttc cgcaggagag ctccagaagg gtaacgagct tgctttgccg tccaagtatg 6300

tgaacttcct ctatctggca tcccactacg agaagctcaa gggcagccca gaggataacg 6360

aacagaagca actgtttgtg gagcaacaca agcattatct tgacgagatc attgaacaga 6420

tttcggagtt cagtaagcgc gtcatcctcg ccgacgcgaa tttggataag gttctctcag 6480

cctacaacaa gcaccgggac aagcctatca gagagcaggc ggaaaatatc attcatctct 6540

tcaccctgac aaaccttggg gctcccgctg cattcaagta ttttgacact acgattgatc 6600

ggaagagata cacttctacg aaggaggtgc tggatgcaac ccttatccac caatcgatta 6660

ctggcctcta cgagacgcgg atcgacttga gtcagctcgg gggggataag agaccagcgg 6720

caaccaagaa ggcaggacaa gcgaagaaga agaagtagca attcggtacg ctgaaatcac 6780

cagtctctct ctacaaatct atctctctct attttctcca taaataatgt gtgagtagtt 6840

tcccgataag ggaaattagg gttcttatag ggtttcgctc atgtgttgag catataagaa 6900

acccttagta tgtatttgta tttgtaaaat acttctatca ataaaatttc taattcctaa 6960

aaccaaaatc cagtactaaa atccagatct cctaaagtcc ctatagatct ttgtcgtgaa 7020

tataaaccag acacgagacg actaaacctg gagcccagac gccgttcgaa gctagaagta 7080

ccgcttaggc aggaggccgt tagggaaaag atgctaaggc agggttggtt acgttgactc 7140

ccccgtaggt ttggtttaaa tatgatgaag tggacggaag gaaggaggaa gacaaggaag 7200

gataaggttg caggccctgt gcaaggtaag aagatggaaa tttgatagag gtacgctact 7260

atacttatac tatacgctaa gggaatgctt gtatttatac cctatacccc ctaataaccc 7320

cttatcaatt taagaaataa tccgcataag cccccgctta aaaattggta tcagagccat 7380

gaataggtct atgaccaaaa ctcaagagga taaaacctca ccaaaatacg aaagagttct 7440

taactctaaa gataaaagat ctttcaagat caaaactaga gtcgacctgc aggcatgcaa 7500

gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 7560

ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 7620

ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgctagagc agcttgagct 7680

tggatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt 7740

ggcgggtaaa cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg 7800

aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga 7860

tcaaagtact ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt 7920

gcagccgtct tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc 7980

cctgcccttt tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg 8040

cgactagaac cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta 8100

tgcccgcgtc agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc 8160

cggctgcacc aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct 8220

ggccaggatg cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg 8280

cctggcccgc agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc 8340

gggcctgcgt agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt 8400

gttgaccgtg ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg 8460

gagcgggcgc gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac 8520

cccggcacag atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga 8580

ggcggctgca ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga 8640

ggaagtgacg cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga 8700

ggccgacgcc ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac 8760

caggacggcc aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg 8820

gccgggtacg tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg 8880

gccggtttgt ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc 8940

gagcgccgcc gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc 9000

gctgcgtata tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta 9060

tcgctgtact taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc 9120

gcgccctgca actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg 9180

cccgcgattg ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc 9240

cgacgattga ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag 9300

cgccccaggc ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc 9360

cggtgcagcc aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc 9420

agcgcattga ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca 9480

aaggcacgcg catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc 9540

ttgagtcccg tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg 9600

ttcttgaatc agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa 9660

ttaaatcaaa actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac 9720

gctaagtgcc ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc 9780

agacacgcca gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct 9840

gaagatgtac gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc 9900

gcagctacca gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag 9960

gaggcggcat ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg 10020

gaggaacggg cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca 10080

ctggaacccc caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa 10140

atcggcgcgg cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag 10200

cggcaacgca tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga 10260

atccgcaaag aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc 10320

aagggcgacg agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat 10380

agtcgcagca tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc 10440

gaggtgatcc gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc 10500

atggccagtg tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc 10560

atgaaccgat accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt 10620

gcggacgtac tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta 10680

gaaacctgca ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag 10740

aacggccgcc tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta 10800

aagagcgaaa ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc 10860

gagatcacag aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc 10920

gatcccggca tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa 10980

gccagatggt tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag 11040

ttctgtttca ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag 11100

gaggaggcgg ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc 11160

gaagcatccg ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg 11220

gaaaaaggtc gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg 11280

tacattggga accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca 11340

cacatgtaag tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta 11400

aaacttatta aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca 11460

gccgaagagc tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct 11520

tcgcgtcggc ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat 11580

ctaccagggc gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc 11640

accctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 11700

gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 11760

agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt 11820

gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 11880

tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc 11940

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 12000

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 12060

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 12120

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 12180

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 12240

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 12300

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 12360

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 12420

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 12480

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 12540

tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 12600

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 12660

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 12720

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct 12780

aggtactaaa acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg 12840

atccccagta agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc 12900

gaccggacgc agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca 12960

ataaagccac ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg 13020

gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga 13080

tctttaaatg gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc 13140

agtaagtaat ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat 13200

atgtcgatgg agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg 13260

ctttgttcat cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga 13320

ttgctccagc catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc 13380

agccatagca tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg 13440

tccgtcattt ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga 13500

gacattcctt ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt 13560

gatattctca ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac 13620

cccaagaagc taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc 13680

ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat 13740

cgacggagcc gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca 13800

atcaacatgc taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt 13860

tcttccgaat agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct 13920

gacgccgtcc cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg 13980

gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct 14040

tagacaactt aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat 14100

tcgggggatc tggattttag tactggattt tggttttagg aattagaaat tttattgata 14160

gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg 14220

aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa 14280

actcgagctt gtcgatcgac agatccggtc ggcatctact ctatttcttt gccctcggac 14340

gagtgctggg gcgtcggttt ccactatcgg cgagtacttc tacacagcca tcggtccaga 14400

cggccgcgct tctgcgggcg atttgtgtac gcccgacagt cccggctccg gatcggacga 14460

ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa attgccgtca accaagctct 14520

gatagagttg gtcaagacca atgcggagca tatacgcccg gagtcgtggc gatcctgcaa 14580

gctccggatg cctccgctcg aagtagcgcg tctgctgctc catacaagcc aaccacggcc 14640

tccagaagaa gatgttggcg acctcgtatt gggaatcccc gaacatcgcc tcgctccagt 14700

caatgaccgc tgttatgcgg ccattgtccg tcaggacatt gttggagccg aaatccgcgt 14760

gcacgaggtg ccggacttcg gggcagtcct cggcccaaag catcagctca tcgagagcct 14820

gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg ccagtgatac acatggggat 14880

cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg accgattcct tgcggtccga 14940

atgggccgaa cccgctcgtc tggctaagat cggccgcagc gatcgcatcc atagcctccg 15000

cgaccggttg tagaacagcg ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct 15060

gtgcacggcg ggagatgcaa taggtcaggc tctcgctaaa ctccccaatg tcaagcactt 15120

ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac ataacgatct ttgtagaaac 15180

catcggcgca gctatttacc cgcaggacat atccacgccc tcctacatcg aagctgaaag 15240

cacgagattc ttcgccctcc gagagctgca tcaggtcgga gacgctgtcg aacttttcga 15300

tcagaaactt ctcgacagac gtcgcggtga gttcaggctt tttcatatct cattgccccc 15360

cgggatctgc gaaagctcga gagagataga tttgtagaga gagactggtg atttcagcgt 15420

gtcctctcca aatgaaatga acttccttat atagaggaag gtcttgcgaa ggatagtggg 15480

attgtgcgtc atcccttacg tcagtggaga tatcacatca atccacttgc tttgaagacg 15540

tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac 15600

cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga tggcatttgt 15660

aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct gggcaatgga 15720

atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc ctttggtctt 15780

ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgttat 15840

cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 15900

tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 15960

ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 16020

tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 16080

gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 16140

cgagagtgtc gtgctccacc atgttggcaa gctgctctag ccaatacgca aaccgcctct 16200

ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 16260

gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 16320

acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 16380

aggaaacagc tatgaccatg attacg 16406

Claims (4)

1. Application of pC1300-MAS-Cas9 gene editing system in 84K poplar gene editing, comprising the following steps:

   step (1): selection of PagPDS Gene target sites

   According to the 84K Yang Jiyin sequence, a CDS region of the PagPDS gene is found, the structure is analyzed, a potential Cas9 target site is searched, and gRNA is designed according to the position and GC content of the target site;

   in the step (1), pagPDS gene, pagPDS form P. Glandulosa sequence is shown as sequence 1 in a sequence table, pagPDS form P. Alba sequence is shown as sequence 2 in the sequence table;

   step (2): design of gRNA

   According to the design principle of PAM sequences, 3 gRNAs, namely gRNA1, gRNA2 and gRNA3, are designed in sequences without SNP loci, and see sequences 9-11 in a sequence table;

   step (3): construction of CRISPR/Cas9 Gene knockout vector

   1) Annealing polymerization of oligonucleotides

   Based on the subsequently ligated vector adaptor sequences, desalted oligonucleotide strands with specific adaptors are designed, and the synthesized oligonucleotide primers are primed with ddH ₂ Diluting with water; annealing reaction system: 1. Mu.L of sense oligonucleotide, 1. Mu.L of antisense oligonucleotide, add ddH ₂ 0 to 50 mu L, uniformly mixing the prepared annealing reaction buffer solution, centrifuging briefly, then using a PCR instrument, standing at 95 ℃ for 5min at room temperature for 20min, mixing the primers, performing deformation annealing to form an oligonucleotide fragment with a sticky end, and immediately using the oligonucleotide fragment or standing at-20 ℃ for long-term storage after the standing at room temperature is finished;

   2) Construction of a Single intermediate vector

   Inserting the gRNA sequence into a carrier containing SK-gRNA, wherein the SK-gRNA sequence is shown as a sequence 12 in a sequence table; ligating gRNA1, gRNA2, gRNA3 into SK-gRNA by AarI enzyme;

   3) Multiple intermediate support polymerization

   Polymerizing 3 SK-gRNA intermediate vectors by utilizing the property that BamHI and BglII are isotail enzymes, performing enzyme digestion by using KpnI and BamHI as vectors to provide segmented SK-gRNA1, and performing enzyme digestion by using KpnI+SalI; SK-gRNA2 was digested with XhoI+XbaI; SK-gRNA3 was digested with Spel+BgI II;

   4) Construction to final vector

   The pC1300-Cas9 vector is digested with KpnI and BamHI, SK-gRNA is digested with KpnI and BglII and fragments are recovered and ligated to the pC1300-Cas9 vector using a final vector ligation system;

   the final vector pC1300-Cas9 was digested with KpnI and BamH I and recovered; three intermediate vectors were simultaneously ligated to final vector pC1300-Cas9 with KpnI and BamH I cleavage sites, which were started by the 2x35S and MAS promoters, respectively;

   5) Transformation competence

   After transformation, taking 100 mul of coated plates, selecting 10 monoclone on an LB+Kan plate the next day, adding LB+Kan into a 1.5mL centrifuge tube, shaking bacteria in a shaking table at 37 ℃ and 250rmp, and performing PCR detection and sequencing after the bacteria are well shaken;

   step (4): genetic transformation of poplar

   Step (5): acquisition and statistics of transgenic plants

   Respectively obtaining 40 PagPDS-2x35S-Cas9 and PagPDS-MAS-Cas9 transgenic plants by an agrobacterium transformation leaf disc method, wherein the transgenic plants of the two vectors have albino phenotype; among the 40 transgenic plants in PagPDS-2x35S-Cas9, albino seedlings are 27, and mutation rate is 67.5%, wherein 21 plants are of a pure-whitening phenotype, and 6 plants are of a yellow-green phenotype; among the 40 transgenic plants in PagPDS-MAS-Cas9, albino seedlings are 30, wherein 23 plants are pure white standard type, and 7 plants are yellow-green phenotype;

   step (6): directional editing of poplar genes by CRISPR system

   1) Identification of transgenic plant DNA level

   After the leaves are subjected to agrobacteria dip-dyeing, screening and growing in a screening culture medium containing Hyg, and identifying rooting resistant seedlings after the material is subjected to differentiation, elongation and rooting processes;

   2) Mutation identification of transgenic plants

   After obtaining positive transgenic plants, using specific primers for gRNA target site detection, carrying out PCR amplification to obtain DNA sequence fragments with potential target site modification, then connecting the fragments to a T carrier after electrophoresis separation, converting escherichia coli to obtain single colonies, extracting plasmids for sequencing of target sites, comparing sequencing results with a control sequence, and analyzing mutation events.
2. 2. The use of the pC1300-MAS-Cas9 gene editing system according to claim 1 in 84K poplar gene editing, characterized in that:

   in step (3) 2), the specific steps are as follows: firstly, carrying out Aar I enzyme digestion on SK-gRNA to form a carrier with a sticky end, carrying out denaturation annealing on the gRNA after mixing to form a fragment with the sticky end, connecting the carrier and the fragment, and converting DH5

   

   The ligation plasmid was obtained and sequenced.
3. 3. Use of the pC1300-MAS-Cas9 gene editing system according to claim 2 in 84K poplar gene editing, characterized in that: the step (4) is specifically as follows:

   1) Explants as transformation material: taking stem segments and tender leaf blades of 84 poplar growing for 3-4 weeks as explant transformation materials, cleaning the blades with clean water after taking materials, sterilizing in an ultra clean bench with 20% sodium hypochlorite for 20min, cleaning with sterile distilled water for at least 5 times, ensuring that no sodium hypochlorite remains on the surface of the material, and sucking redundant distilled water with sterile filter paper;

   2) Tissue culture seedlings are used as transformation materials: cutting off plant leaves in an ultra-clean bench by scissors, taking small discs with obvious main veins by using a sterile 6mm puncher, and putting the small discs into a normal culture medium for standby so as to prevent water dispersion;

   3) Taking out the stored carrier agrobacterium bacterial liquid from a refrigerator with ultralow temperature of minus 80 ℃ in a laboratory, placing the carrier agrobacterium bacterial liquid on ice for melting, using a sterilized inoculating needle, taking a small amount of bacterial liquid, dividing the bacterial liquid on a prepared YEB solid culture medium with the concentration of 100mg/L Kan and 50mg/LRif, sealing the solid culture medium, inversely placing the solid culture medium in a 28 ℃ incubator for culturing for 48 hours, picking single bacterial colonies after single bacterial colonies grow out, shaking the bacterial colonies overnight in an LB liquid culture medium with the concentration of 100mg/L Kan and 50mg/L Rif, and keeping the bacterial liquid to amplify in a logarithmic phase;

   4) Pouring the bacterial liquid into a sterilized centrifugal bottle, centrifuging for 30min, discarding the upper bacterial liquid, then suspending the precipitate by using the prepared suspension, wherein the OD600 = 0.3-0.4;

   5) Culturing the suspended bacterial liquid in a shaking table with the temperature of 50-1000rmp for 1h;

   6) Soaking the explant or tissue culture material in the suspended bacterial liquid for dip-dyeing: shaking in a 50rmp shaking table for 1-2h at room temperature;

   7) After the dip dyeing is finished, sucking redundant dip dyeing liquid into the leaf by using sterile filter paper, transferring the leaf into a differentiation culture medium, and culturing in a dark inversion mode for 2d, wherein the optimal temperature is 19-21 ℃;

   8) After two days, the agrobacterium grows out of the periphery of the dip-dyed material, the material is required to be cleaned, the material is cleaned by sterile water for four times, the filtered washing liquid is used for cleaning once, and after the washing liquid is added, the material is placed in a shaking table with 50-1000rmp for cleaning for 1h;

   9) Placing the cleaned material in a differentiation medium containing 500 mg/Lcefotam and 2mg/L Hyg of antibiotics, culturing in dark for 2 weeks, and culturing under light for two weeks;

   10 After callus growth, transferring the material to an elongation culture medium containing 50mg/L Hyg, and replacing the culture medium every 2-4 weeks;

   11 When the material height is 2cm, transferring the material into rooting culture medium containing 2mg/L Hyg, and the whole transformation process is 3-8 months.
4. 4. The use of the pC1300-MAS-Cas9 gene editing system of claim 3 in 84K poplar gene editing, characterized in that: step (6) 2) is specifically as follows:

   sanger sequencing was performed on the transgenic plant extract DNA, pagPDS-2x35S-Cas9 at the T1 site, and 53.75% of the mutations in all albino phenotype plants were single nucleotide insertions, and the rate of detection of at least one allelic mutation was 65% in 26 edited plants; 21 strains with simultaneous mutations of two alleles, including 20 homozygous mutations and 1 biallelic mutation; in the T2/T3 locus, the mutation rate was 67.5%, including 21 biallelic mutations and 4 homozygous mutations, and a deletion of 2 to 5nt was found near PAM;

   in the PagPDS-MAS-Cas9 system, 30 plants are albino phenotype, the mutation rate is 75%, the pure albino plants are 23 plants, and the yellow-green phenotype plants are 7 plants; albino phenotype plants had alleles with significant large fragment deletions at T1 and T2/T3; regarding analysis of T1, mutations of at least one allele were detected in 29 plants, with 22 mutations in both alleles, including 19 homozygous mutations and 3 bi-allelic mutations, and a mutation rate of 72.5% at the T2/T3 locus, including 20 homozygous mutations and 2 bi-allelic mutations.

Images