CN112359057B - Application of CRISPR/Cas12a gene editing system in 84K poplar gene editing - Google Patents

Abstract

The invention relates to application of a CRISPR/Cas12a gene editing system in 84K poplar gene editing, belonging to the technical field of plant genetic engineering. Including selection of the target site for the PagPDS gene; constructing a vector; genetic transformation of poplar; obtaining a transgenic plant; and (3) analyzing the directional editing condition of the CRISPR system on the poplar genes. The CRISPR/Cas12a gene editing system disclosed by the invention is applied to 84K poplar gene editing, and hybrid poplar '84K' is used for evaluating the targeted mutation of the CRISPR/Cas12a system; the invention uses three Cas12a nucleases AsCas12a, lbCas12a and FnCas12a, and finally provides a simple and effective tool capable of editing a plurality of woody plant genes.

Description

Application of CRISPR/Cas12a gene editing system in 84K poplar gene editing

Technical Field

The invention relates to application of a CRISPR/Cas12a gene editing system in woody plant gene editing, in particular to application of the CRISPR/Cas12a gene editing system in 84K poplar gene editing, belonging to the technical field of biological gene engineering.

Background

The woody plant has long life, high heterozygosis of genome, perennial secondary growth process and wood formation characteristic. Woody plants are limited by gene editing tools and it is difficult to study gene function by mutants, such as the creation of large numbers of single or multiple gene-deleted mutants.

Poplar is a model plant in woody plants, is an important source of paper, wood, building materials and biofuels, and has high economic and ecological values. For researchers, poplar is an ideal material for studying the activity of perennial secondary vascular stratification, wood formation, and for studying secondary cell walls.

In recent years, the CRISPR/Cas9 genome editing system has been successfully applied to poplar, but Cas12a has not been reported in poplar. Cas12a is a novel Cas endonuclease from Prevotella (Prevotella) and Francisella (Francisella) and has been used as a gene editing system for a variety of plant species, such as arabidopsis, rice, cotton, tobacco, soybean. Cas12a protein has simple structure and small molecular weight, and the mediated gene editing does not need trancrRNA and only needs a short single-stranded crRNA. Cas12a recognizes that the PAM sequence is a 3' end T-rich region (the PAM sequence corresponding to SpCas9 is 5' -NGG-3 '), so that the genome editing mediated by Cas12a can not only enlarge targets except for CRISPR/Cas9, but also generate staggered cutting, generate high-efficiency editing effect on editing gene sites, and introduce insertion or deletion mutation of base sequences with different degrees.

Therefore, how to establish and efficiently apply the CRISPR/Cas12a gene editing system in woody plant 84K poplar gene editing research becomes a technical problem which needs to be solved in the technical field urgently.

Disclosure of Invention

One of the purposes of the present invention is to provide a simple and effective application of a CRISPR/Cas12a gene editing system in 84K poplar gene editing, aiming at the defects existing in the prior art.

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

the application of the CRISPR/Cas12a genome editing system in 84K poplar gene editing comprises the following steps:

step (1): selection of 84K poplar phytoene dehydrogenase gene 8 target site

According to a CTAB method, extracting wild poplar '84K' gene DNA, and cloning phytoene dehydrogenase gene 8 (PagPDS gene) from the extracted poplar '84K' gene DNA;

amplifying PagPDS by using a specific primer sequence F shown in a sequence 3 in a sequence table and a specific primer sequence R shown in a sequence 4 in the sequence table; the PCR products were electrophoresed on a 1% agarose gel; DNA was recovered using the TIANgel Midi purification kit and cloned into the pMD18-T Simple vector;

SNP sites are excluded according to the sequence, and 5 sgRNA targeted gene sites are designed according to GC content and PAM sequence (FnCas 12a, PAM 5'-TTN-3'; asCas12a, PAM 5'-TTTN-3'; lbCas12a, PAM 5 '-TTTN-3');

step (2): construction of vectors

5 gRNAs, namely gRNA1, gRNA2, gRNA3, gRNA4 and gRNA5, are connected in series with tRNA-crRNA to form a crRNA box, wherein the crRNA box comprises a mature direct repeat (DR sequence TAATTTCTACTAAGTGTAGAT) and a gRNA sequence of 24bp and is driven by an arabidopsis RNA polymerase III promoter AtU6-26, a 2x35S promoter in a pCambia1300 binary vector drives three endonuclease AsCas12a, lbCas12a and FnCas12a with different sources, and the constructed vectors are named as PagPDS-AsCas12a, pagPDS-LbCas12a and PagPDS-FnCas12a respectively;

and (3): genetic transformation of poplar

Respectively transferring the three expression vectors constructed in the step (2) into agrobacterium GV3101 by an electric shock method, transferring the vectors into poplar by agrobacterium mediation, and performing the transformation steps as follows: the 84K tissue culture seedlings of the hybrid poplar clone for genetic transformation are cultured under the conditions that the culture temperature is 23-25 ℃, the illumination is 16/8h (day/night) and the illumination intensity is 50 mu Mm-2 s-1;

infecting an 84K leaf disc with Agrobacterium containing the vector at OD600= 0.6-0.8, co-culturing the infected leaf disc on adventitious bud induction medium (SIM, murashige-Skoog (MS) minimal medium supplemented with 0.5 mg/L6-benzyl aminopurine (6-BA, 6-benzylaminopurine) and 0.05mg/L naphthalene acetic acid) under dark conditions at 22 + -2 ℃ for 3 days, transferring the co-cultured leaf disc to SIM containing 3mg/L hygromycin B (hygromycin B) and 200mg/L Timentin (Timentin), inducing and screening resistant adventitious buds under conditions of culture temperature of 23-25 ℃, illumination intensity of 16/8h (day/night), illumination intensity of 50 Mm-2s-1, transferring the cultured leaf disc to 3mg/L NAmgs-2 s-1, and extracting the DNA of the root induction medium containing 3 mg/0.05 mg/L, and performing PCR (PCR) until the root induction medium containing 0.05mg/L of DNA is obtained, and the root induction medium is added with 0.5 mg/L;

and (4): obtaining transgenic plants

30 transgenic plants positive for resistance screening are respectively obtained by a PagPDS-AsCas12a, pagPDS-FnCas12a and PagPDS-LbCas12a leaf disc transformation method through agrobacterium, and biallelic homozygous mutants and heterozygous mutants of PDS show albino phenotypes.

Preferably, after step (4), the following step (5) is added:

and (5): temperature treatment (Heat treatment)

To further improve the editing efficiency of Cas12a, the present inventors heat-treated the callus cultured after agrobacterium infection (callus can be grown by dark culture of the tissue after agrobacterium infection on a medium containing antibiotics and a screening agent at room temperature for about one and a half months).

Preferably, the step (5) is specifically as follows:

firstly, culturing a transformation material in the dark for 2 days, cleaning agrobacterium, transferring the material to an induction callus culture medium, inducing callus in the dark at room temperature (22 ℃) for 4 weeks, then carrying out heat treatment on the callus at 28 ℃, culturing at 28 ℃ for 30h, transferring to 22 ℃ for culturing for 42h, repeating for 4 times, placing the callus at room temperature for normal culture, transferring the material to the induction budding culture medium for induction budding, and recording the morphology and the molecular phenotype.

And (6): directional editing condition analysis of CRISPR system on poplar genes

To analyze mutation of PagPDS in transgenic T0 generation, genomic DNA was extracted from stable transgenic and wild-type plants using CTAB method, endogenous PagPDS fragments were PCR amplified by primer pair sequence F and primer pair sequence R using genomic DNA as template, primers covering five target site regions inside PagPDS gene, PCR products were isolated on 1% agarose gel, recovered and cloned into pMD18-T Simple vector, mutations were identified by Sanger sequencing of single clones, all sequence results were compared to reference sequence of PagPDS by alignment in SnapGene.

Has the advantages that:

the application of the CRISPR/Cas12a gene editing system in woody plant gene editing comprises the steps of evaluating the targeted mutation of the CRISPR/Cas12a system by using hybrid poplar '84K' (Populus alba multiplied by Populus glaandusas); the invention uses three Cas12a nucleases, namely AsCas12a (Acylaminococcus sp.BV3L6), lbCas12a (Lachnospiraceae bacterium ND 2006) and FnCas12a (Francisela tularensis subsp.novicidain U112), and finally provides a simple and effective tool capable of simultaneously targeting and editing a plurality of woody plant genes, and the invention also optimizes the working temperature of Cas12a and improves the editing efficiency of Cas12a; whether the Cas12a has editing capacity in poplar trees or not is researched, and the research on the functional genome of a CRISPR/Cas12 a-mediated polygene editing system in woody plants is facilitated.

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

Drawings

FIG. 1 is an alignment of the sequencing results of the PagPDS gene in example 1 of the present invention.

FIG. 2 is a schematic representation of the PagPDS target sites (T1-T5) and their sequences in example 1 of the invention.

FIG. 3 is a schematic diagram showing the structure of the final vector in example 1 of the present invention.

FIG. 4 is the phenotype of the PagPDS-AsCas12a/LbCas12a/FnCas12a mediated transgenic plants in example 1 of the present invention.

FIG. 5 is a schematic diagram showing the culture conditions of calli under 28 ℃ heat stress in example 1 of the present invention.

FIG. 6 is a schematic diagram of callus under heat stress at room temperature (22 ℃) and 28 ℃ of the callus in example 1 of the present invention.

Fig. 7 is an editing efficiency at room temperature and heat treatment in example 1 of the present invention.

Fig. 8 is a gene sequence for gene recognition of the PAM: cas12a nuclease in example 1 of the present invention.

FIG. 9 is a schematic diagram showing the positions of 5 target leader sequences of PagPDS Locus, a phytoene dehydrogenase gene PagPDS of poplar in example 1 of the present invention.

Detailed Description

Example 1

The application of the CRISPR/Cas12a gene editing system in poplar gene editing comprises the following steps:

step (1): selection of target sites for PagPDS Gene

Extracting wild poplar '84K' genome DNA according to a CTAB method, and cloning a phytoene dehydrogenase gene 8 gene (PagPDS gene) from the extracted poplar '84K' genome DNA, wherein the PagPDS form P.glandulosa sequence is shown as sequence 1 in a sequence table, and the PagPDS form P.alba sequence is shown as sequence 2 in the sequence table;

amplifying PagPDS by using a specific primer sequence F shown as a sequence 3 in a sequence table and a specific primer sequence R shown as a sequence 4 in the sequence table; the PCR products were electrophoresed on a 1% agarose gel; DNA was recovered using a TIANgel Midi purification kit (Tiangen, beijing, china) and cloned into a pMD18-T Simple vector, see sequence 5 in the sequence Listing;

SNP sites were excluded according to GC content and PAM sequence (FnCas 12a, PAM 5'-TTN-3'; asCas12a, PAM 5'-TTTN-3'; lbCas12a, PAM 5 '-TTTN-3');

step (2): construction of vectors

Designing 5 gRNAs (gRNA 1, gRNA2, gRNA3, gRNA4, gRNA 5), as shown in FIG. 2, which are PagPDS target sites (T1-T5) and sequence schematic diagrams thereof in example 1 of the present invention; where blue indicates exon (CDS 1-CDS 4), orange indicates intron, CDS exon regions are schematically depicted: CDS1 is the first exon region; CDS2 is the second exon region; CDS3 is the third exon region; CDS4 is the fourth exon region; the 5 selected target sequences positioned in the exon structure region are called T1, T2, T3, T4 and T5 for short; connecting 5 gRNAs (gRNA 1, gRNA2, gRNA3, gRNA4, gRNA 5) shown as sequences 9-13 in a sequence table and tRNA-crRNA in series, wherein each unit comprises a mature red letter in Direct Repeat (DR) and a gRNA sequence of 24bp, and a plurality of crRNA boxes driven by an Arabidopsis RNA polymerase III promoter AtU6-26, a 2x35S promoter in a pCambia1300 binary vector drives AsCas12a, lbCas12a and FnCas12a, a FnCas12a sequence, an AsCas12a sequence and a LbCas12a sequence which are respectively shown as sequences 6-8 in the sequence table; the constructed vectors are named as PagPDS-AsCas12a, pagPDS-LbCas12a and PagPDS-FnCas12a respectively, and are shown as sequences 14-16 in a sequence table; FIG. 3 is a schematic diagram showing the structure of the final vector in example 1 of the present invention; wherein LB refers to the left boundary; RB refers to the right boundary; DR refers to direct repeats; multi-crRNA expression cassette refers to multiple guide RNA nucleic acid sequence expression cassettes; atU6 means a promoter of Arabidopsis U6 gene; t1, T2, T3, T4, T5 refer to 5 sequences: gRNA1, gRNA2, gRNA3, gRNA4, gRNA5; cas12a cassette refers to Cas12a nuclease expression cassette; 2X35S refers to the tobacco mosaic virus promoter; asCas12a refers to Cas12a nuclease from aminoacetococcus (Acidaminococcus); lbCas12a refers to a Cas12a nuclease derived from Lachnospiraceae (Lachnospiraceae bacteria); fnCas12a refers to Cas12a nuclease from Francisella novicida; TERM refers to terminator;

AtU6-DR-guide1-DR-guide2-DR-guide3-DR-guide4-DR-guide5-poly T: crRNA expression cassettes (crRNA sequence, see sequence 17 in the sequence listing) for AsCas12a, lbCas12a and FnCas12a multiple gene editing vectors;

and (3): genetic transformation of poplar

The expression vector is transferred into agrobacterium GV3101 by an electric shock method, and the vector is transferred into poplar by agrobacterium mediation, and the transformation steps are as follows: the hybrid poplar clone 84K tissue culture seedling for genetic transformation is cultured under the conditions that the culture temperature is 23-25 ℃, the illumination is 16/8h (day/night) and the illumination intensity is 50 mu Mm-2 s-1;

the agrobacterium containing the vector infects an 84K leaf disc when OD600= 0.6-0.8, the infected leaf disc is co-cultured on an adventitious bud induction medium (SIM, murashige-Skoog (MS) basic medium added with 0.5 mg/L6-benzyl aminopurine (6-BA, 6-benzylaminopurine) and 0.05mg/L naphtaleneacetic acid (NAA, naphthylacetic acid)) under a dark condition at a temperature of 22 +/-2 ℃ for 3 days, the co-cultured leaf disc is transferred to the SIM containing 3mg/L hygromycin B and 200mg/L Timentin (Timentin) to induce and screen adventitious buds under conditions of a culture temperature of 23-25 ℃, a light intensity of 16/8h (day/night), a light intensity of 50 mu Mm-2s-1, the induced buds are transferred to a medium containing 3 mg/L-2 s-1, the induced buds are transferred to a basic medium containing 3 mg/0.6 mg/L DNA and PCR is extracted until the DNA is added to the rooting medium, and the rooting medium is added with 0.05mg/L of DNA and 0.05mg/L of the rooting medium until the rooting medium is screened out, and the rooting medium is added with about 30 days;

and (4): obtaining transgenic plants

30 transgenic plants are obtained by agrobacterium tumefaciens transformation leaf disc method through PagPDS-AsCas12a, pagPDS-LbCas12a and PagPDS-FnCas12a, wherein biallelic homozygous and heterozygous mutant of PDS show albinism phenotype, as shown in figure 4, the phenotype of the PagPDS-AsCas12a/LbCas12a/FnCas12a mediated transgenic plants in the embodiment 1 of the invention; wherein PagPDS-AsCas12a is a transgenic poplar with 5 target guide sequences of phytoene dehydrogenase gene PagPDS of poplar and a gene editing vector of Cas12a nuclease derived from aminoacidococcus; the PagPDS-LbCas12a is a transgenic poplar with 5 target guide sequences of the phytoene dehydrogenase gene PagPDS of the poplar and a gene editing vector of Cas12a nuclease from Lachnospiraceae (Lachnospiraceae); the PagPDS-FnCas12a is a transgenic poplar with 5 target guide sequences of a phytoene dehydrogenase gene PagPDS of the poplar and a gene editing vector of Cas12a nuclease derived from Francisella novicida (Francisella novicida); albisim refers to albino seedlings; pale green refers to light green; green is Green;

in all three Cas12a transgenic poplars, albino phenotype plants were observed, which means that mutations were generated at the PDS gene, and all the three Cas12a systems had gene editing ability in poplars, the proportion of albino seedlings indicated the efficiency of gene editing, the editing efficiency in the AsCas12a system was 56.7%, in the LbCas12a system was 30%, and in the FnCas12a system was only 3.4%; see table 1;

TABLE 1 mutations in T0 generation Populus trees generated by CRISPR/Cas12a System at Room temperature

And (5): temperature treatment (Heat treatment)

To further improve the editing efficiency of Cas12a, the inventors heat-treated the callus cultured after agrobacterium infection: firstly, culturing a transformation material in the dark for 2 days, cleaning agrobacterium, transferring the material to an induction callus culture medium, inducing callus in the dark at room temperature (22 ℃) for 4 weeks, then, carrying out heat treatment on the callus at 28 ℃, culturing at 28 ℃ for 30h, transferring to 22 ℃ for culturing for 42h, repeating for 4 times, placing the callus at room temperature for normal culture, transferring the material to an induction budding culture medium for induction budding, and recording the morphology and molecular phenotype;

as shown in FIG. 5, it is a schematic view of the culture conditions of callus under heat stress at 28 ℃ in example 1 of the present invention; wherein, in the middle of fig. 5: the method comprises the following steps: dark for dark, room temperature for Room temperature; below in fig. 5: co-culture is Co-culture, callus induced is induced Callus, heat stress is Heat shock treatment, and Bud induced is Bud induction; the top of fig. 5 is, from left to right: 2day for 2 days, 4week for 4 weeks, 12day for 12 days, 2month for 2 months;

FIG. 6 is a schematic diagram showing callus of example 1 under heat stress at room temperature (22 ℃) and 28 ℃; wherein Heat stress represents Heat shock treatment; room temperature refers to Room temperature; pagPDS-AsCas12a refers to transgenic callus with 5 target guide sequences of the phytoene dehydrogenase gene PagPDS of poplar and a gene editing vector of Cas12a nuclease from aminoacidococcus (aminoacidococcus); pagPDS-LbCas12a refers to transgenic callus with 5 target guide sequences of poplar phytoene dehydrogenase gene PagPDS and a gene editing vector of Cas12a nuclease from the family trichospiraceae (Lachnospiraceae); pagPDS-FnCas12a refers to transgenic callus with 5 target guide sequences of the phytoene dehydrogenase gene PagPDS of poplar and a gene editing vector of Cas12a nuclease from Francisella novicida;

more shoots with albino phenotype induced after heat treatment than untreated induced shoots; the gene editing efficiency of the AsCas12a system is still the highest, and is up to 70%, the gene editing efficiency of LbCas12a is 33%, and the gene editing efficiency of FnCas12a is only 6.7%; phenotypic statistics are shown in table 2 below;

table 2: mutations in T0 generation poplars generated by CRISPR/Cas12a system after heat shock treatment

Compared with room temperature, the editing efficiency of the PagPDS-AsCas12a after heat shock treatment is improved by 13.3%, the editing efficiency of the PagPDS-LbCas12a is improved by 3%, and the editing efficiency of the PagPDS-FnCas12a is improved by 3.3%;

and (6): directional editing of 84K poplar gene by CRISPR system

To analyze mutation of PagPDS in transgenic T0 generation, endogenous PagPDS fragments were PCR amplified using CTAB method, extracting genomic DNA from stable transgenic and wild type plants, using genomic DNA as template, using primer pair sequence F, see sequence 18 in the sequence table, and primer pair sequence R, see sequence 19 in the sequence table, primers covering five target site regions containing Cas12a system, PCR products were separated on 1% agarose gel, recovered and cloned into pMD18-T Simple vector, mutations were identified by Sanger sequencing of single clones, all sequence results were compared to reference sequence of PagPDS by alignment in SnapGene, results are shown in fig. 1, which is the PagPDS gene sequencing result alignment in example 1 of the present invention, wherein blue color represents exon; the red horizontal line represents the PAM sequence; bright yellow represents the gRNA site; and comparing the sequencing result with the sequences of 5 gRNAs, wherein if the gRNA position of the sequencing result is cut by nuclease, the sequence of the gRNA is changed and is inconsistent with the sequence in the original genome, and the sequence is generally expressed as deletion, mutation or addition of a new base sequence.

As shown in FIG. 7, which is the editing efficiency at room temperature and heat treatment in example 1 of the present invention, pagPDS-AsCas12a refers to a transgenic poplar with 5 target guide sequences of phytoene dehydrogenase gene PagPDS of poplar and a gene editing vector of Cas12a nuclease derived from amino acid coccus (Acylaminococcus); pagPDS-LbCas12a refers to a transgenic poplar with 5 target guide sequences of the phytoene dehydrogenase gene PagPDS of poplar and a gene editing vector of Cas12a nuclease derived from pilospiraceae (Lachnospiraceae bacteria); pagPDS-FnCas12a refers to a transgenic poplar with 5 target guide sequences of the phytoene dehydrogenase gene PagPDS of the poplar and a gene editing vector of Cas12a nuclease derived from Francisella novicida (Francisella novicida); heat stress represents Heat shock treatment; room temperature means Room temperature; the vertical coordinate phenotypefrequency refers to the phenotypic frequency; as can be seen from figure 7, the data are mean ± SD (n =3 experiments), each experiment included three biological replicates, asterisks indicate significant differences (./P < 0.05);

and (7): editing event analysis

In order to evaluate the simultaneous editing of multiple target sites, the genomic DNA of the heat-treated transgenic plants is extracted, and the target sequences are amplified by PCR for sequencing, and all mutations generated by Cas12a at 4 targets (T1-T4) are deletions (6-30 nt); however, no mutation was detected at T5, possibly indicating that the last DR may not be completely processed during crRNA maturation; in the PagPDS-AsCas12 a-mediated transgenic plant, alleles carry mutations at three or four target sites, and a large fragment of some alleles is deleted between two target sites, which indicates that the AsCas12a can edit multiple sites in the T0 generation of poplar;

as shown in fig. 8, is a gRNA gene sequence recognized by the gene of Cas12a nuclease PAM in example 1 of the present invention; is the case of insertions and deletions in T0 plants, where the PAM sequence is marked with a horizontal line, the blue letters indicate the target site, and the red letters indicate the insertion; "-" indicates large fragment deletion; "-" indicates a small deletion; the numbers on the right refer to the number of clones detected with such mutant alleles; target1 refers to leader sequence 1; target2 directs leader sequence 2; target3 refers to leader sequence 3; target4 refers to leader sequence 4; target5 directs leader sequence 5;

FIG. 9 is a schematic diagram showing the positions of 5 target leader sequences of PagPDS Locus, a gene PagPDS of phytoene dehydrogenase of poplar in example 1 of the present invention; wherein, T1 directs leader sequence 1; t2 guide sequence 2; t3 guide sequence 3; t4 guide sequence 4; t5 guide sequence 5; delete refers to deletion; is a large fragment deletion mutation of PagPDS Locus; in the process of DNA breaking mediated by Cas12a and repairing, sequences of target sites 1-3, 1-4 and 2-3 are lost;

in the PagPDS-AsCas12a mediated transgenic line, gene editing is diverse; in the examples of the present invention, large fragment deletions between target sites were detected; the deletion frequency of the large fragment is as high as 60 percent; although the editing efficiency of LbCas12a is lower compared to AsCas12a, the edited allele also includes multiple sites and large fragment deletions; of the PagPDS-FnCas12 a-mediated transgenic plants, only two plants with heterozygous alleles exhibited the albino phenotype, indicating that the editing efficiency of FnCas12a is very low.

With respect to the analysis of T1, a mutation (70%) of at least one allele was detected in 21 transgenic plants with a gene editing event (30 positive for antibiotic selection and 21 positive for gene editing), wherein 20 mutations were present in both alleles, including 9 homozygous mutations and 11 biallelic mutations; at T2, the mutation rate was the same as T1 (70%), including 10 homozygous mutations and 10 biallelic mutations; the mutation rate of T3 is low (56.7), the homozygous mutation is 13, and the biallelic mutation is 3 plants; at T4, the mutation rate was lowest (40%), including 13 homozygous mutations and 6 biallelic mutations (table 3); in the CRISPR/Cas12a system of the invention, no mutation was detected at T5, which may indicate that the last DR may not complete processing during crRNA maturation; the results show that AsCas12a can edit multiple sites simultaneously in the T0 generation of poplar.

Table 3: editing situation of each site

According to the invention, a CRISPR/Cas12a system is established and optimized in poplar, and available genome editing tools of woody plants are widened; the editing efficiency of different CRISPR Cas12a systems in poplar trees is tested, and the following results are found: the AsCas12a system is the most efficient system; researches show that the high-efficiency multi-site editing can be carried out in woody plants by using the modified CRISPR/Cas12a and a simple short DR guide sequence (the innovation point of the invention); theoretically, this simple system can be used to edit unlimited genes in poplar simultaneously; thus, a powerful tool is provided for multi-site editing to target members of a multi-gene family.

Poplar callus was generally best cultured at 22 ℃, and in the present study, it was observed that: increasing temperature increases efficiency of Cas12a gene editing; in the AsCas12a system, the callus editing efficiency after heat treatment can be improved from 56.7% to 70%; at 28 ℃, the influence on AsCas12a is strong, the influence on LbCas12a is moderate, and the influence on FnCas12a by temperature is small; the present invention also observes that: no new albino phenotype plants were induced in the calli cultured for two weeks at 34 ℃ and 37 ℃; at these temperatures, callus development was poor due to higher culture temperatures, therefore, the present invention hypothesizes that Cas12a function may depend on the state and temperature of the plant cell.

The result of the invention shows that Cas12a can be used in a woody plant genome editing system; cas12a is a novel CRISPR effector protein, has many features different from Cas9, overcoming some limitations of CRISPR/Cas9 application; the CRISPR/Cas12a not only provides a new alternative method for directed mutagenesis in woody plants, but also greatly improves the range and the precision of genome editing of the woody plants.

Sequence listing

<110> Zhejiang university

Application of <120> CRISPR/Cas12a gene editing system in 84K poplar gene editing

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1716

<212> DNA

<213> phytoene dehydrogenase (PagPDS-A)

<400> 1

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gcattgaact tgagctggca tagtaaatca ttagactctc aagttgcctt gagatgtggc 120

gcttatccta cttgttctca ccaaacgaat gcactagctt ttagaggcag tgaatcaatg 180

ggccattctt tgaaattccc atttggaaat tcttctgcta aaacaagact aaggaatcat 240

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tcaatacgtg aaaatttggg gtgatctttt gtctacgctg taggttgtct gtatggacta 360

tccaagaccg gaccttgata acacggtgaa tttcttagag gctgccttgt tatcttcatc 420

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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

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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

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<213> phytoene dehydrogenase (PagPDS-G)

<400> 2

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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> 24

<212> DNA

<213> Artificial sequence (specific primer F)

<400> 3

gtttgcaggg ctgttgttac agtt 24

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence (specific primer R)

<400> 4

catttaatgg tgcagggaga acttcag 27

<210> 5

<211> 2693

<212> DNA

<213> Artificial sequence (pMD 18-TSimple vector)

<400> 5

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa agaagcatga cggcaagtgg 420

acgattatct ccagaggatc gccgggaacc gaggacgagt tcgtaatcat ggtcatagct 480

gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat 540

aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc 600

actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 660

cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 720

gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 780

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 840

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 900

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 960

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 1020

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 1080

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 1140

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 1200

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 1260

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 1320

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 1380

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 1440

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 1500

gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 1560

ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 1620

ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 1680

tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 1740

accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 1800

atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 1860

cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 1920

tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 1980

tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 2040

gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 2100

agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 2160

aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 2220

gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 2280

tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 2340

gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 2400

tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 2460

aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 2520

catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 2580

acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 2640

tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc 2693

<210> 6

<211> 5566

<212> DNA

<213> Artificial sequence (FnCas 12 a)

<400> 6

gtacccctac tccaaaaatg tcaaagatac agtctcagaa gaccaaaggg ctattgagac 60

ttttcaacaa agggtaattt cgggaaacct cctcggattc cattgcccag ctatctgtca 120

cttcatcgaa aggacagtag aaaaggaagg tggctcctac aaatgccatc attgcgataa 180

aggaaaggct atcattcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 240

cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 300

atgtgacatc tccactgacg taagggatga cgcacaatcc cacccctact ccaaaaatgt 360

caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatttc 420

gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcgaaa ggacagtaga 480

aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcta tcattcaaga 540

tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 600

agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgacatct ccactgacgt 660

aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat aaggaagttc 720

atttcatttg gagaggacag cccaagcttg tcgacatggc ccctaagaag aagagaaagg 780

tcggtattca cggcgttcct gcggcgatgt ccatatatca agagtttgtg aataagtatt 840

cactgagtaa gacactcagg ttcgagctca tccctcaggg caaaaccctt gagaacatca 900

aggctcgggg cctcatactg gatgacgaaa agcgcgccaa agactacaag aaggcgaagc 960

agattatcga caagtatcac cagttcttta ttgaggagat cctctcttca gtctgcatct 1020

ctgaggatct cctgcaaaac tatagtgatg tctacttcaa gctcaagaag agcgatgatg 1080

ataacctcca gaaagatttc aagtcagcca aagacacgat taaaaaacag atctccgaat 1140

acatcaagga ttccgagaag tttaaaaatc tgttcaacca aaatcttatt gacgcgaaaa 1200

agggccagga atcagacttg atcctgtggc tcaagcaatc caaggacaat ggcatcgagc 1260

tgtttaaagc caattccgat attaccgata ttgatgaagc cttggaaatc ataaagtctt 1320

ttaagggttg gactacgtac ttcaaggggt ttcacgagaa tcgcaagaac gtttattcaa 1380

gtaatgacat acccacatct attatctaca gaattgtgga tgacaatctg cccaaattct 1440

tggaaaataa agccaagtat gaatcgctta aggacaaggc tccggaggcc atcaactatg 1500

aacaaataaa gaaggatctg gctgaagagc tgacctttga tatagattac aagacctcgg 1560

aagtgaatca gcgggtgttc agcctcgatg aggtatttga aatagcaaac tttaacaact 1620

acctcaacca atccggcatt accaaattta acactattat tggcggtaaa tttgtgaatg 1680

gggagaacac gaagaggaaa ggcattaacg aatatatcaa cctttacagc cagcagatca 1740

atgataagac cctcaagaag tacaaaatga gtgtcctctt caagcagatc ctcagtgaca 1800

ccgagagcaa aagtttcgta atagataagc ttgaggatga ttccgacgta gtgactacaa 1860

tgcagagttt ttatgaacag atagctgcgt tcaagaccgt ggaagaaaaa tcgataaaag 1920

agacactcag ccttctgttc gacgacctga aggcgcaaaa acttgatctc agcaagatct 1980

attttaagaa cgataagtcc ctgacagatc tgtcgcagca ggtgtttgat gactacagcg 2040

ttatcggcac cgcagtgctg gagtatataa cccaacagat cgcaccgaaa aacctcgaca 2100

atccaagcaa gaaggaacaa gaacttatcg ccaagaagac agagaaggcc aaatatttga 2160

gcctcgagac gattaagttg gcgctggaag agttcaacaa gcatcgggac attgacaagc 2220

agtgcaggtt cgaggagatt ttggcgaact tcgccgcgat cccgatgatt ttcgacgaga 2280

tcgctcagaa caaagacaac ctcgcccaaa tctcgatcaa gtaccagaat caggggaaga 2340

aagatctgct ccaagcctcg gccgaagatg atgtcaaagc gataaaagac ctcctcgacc 2400

agactaacaa cctcttgcat aagctgaaga tttttcacat cagccaatcc gaggataaag 2460

ctaacatact tgacaaggac gagcatttct acttggtctt cgaggagtgc tactttgagc 2520

tggctaatat agtccctttg tacaataaaa tacggaacta tattacccaa aaaccgtatt 2580

ctgatgaaaa attcaagctc aacttcgaaa actccacgct ggccaacgga tgggataaga 2640

acaaagaacc ggacaatacg gccatacttt tcataaagga cgacaagtat tatctcggcg 2700

tcatgaacaa gaagaacaat aaaattttcg acgacaaggc gatcaaagaa aataagggcg 2760

aaggctataa gaagatcgtg tacaagctgc ttccgggcgc aaacaagatg ctcccgaaag 2820

tatttttcag cgccaagtcg attaaatttt acaaccccag cgaggatatt cttcgtatcc 2880

gcaaccatag tactcacact aagaacggtt cgcctcagaa ggggtacgag aaatttgaat 2940

tcaacattga ggactgtcgc aagttcattg atttttacaa gcagtcgata tcgaaacatc 3000

cggagtggaa ggactttggc ttccggttca gtgataccca aaggtacaac tcgattgatg 3060

agttttacag ggaggtcgag aatcagggat ataagctcac gttcgaaaac atctcggagt 3120

catacatcga ttcagtggtc aaccaaggga aactttattt gtttcaaatt tacaacaaag 3180

atttttcagc gtactcgaag ggccgcccaa accttcatac tctgtattgg aaagcgctct 3240

tcgacgagag gaatctccaa gacgtcgtgt ataagctgaa cggagaagct gaactctttt 3300

atcgcaagca atccatccct aagaagataa cccacccagc aaaggaggcc atagccaata 3360

agaataagga caacccgaaa aaggagagcg tgtttgaata cgacctcatt aaggacaaac 3420

ggttcaccga ggataagttt ttttttcact gccctattac cattaacttc aaaagctccg 3480

gcgcgaacaa atttaacgat gaaattaacc tcctgcttaa agagaaggcc aatgatgtgc 3540

acattttgtc tattgataga ggggaaaggc atcttgcata ctatacactc gtggacggta 3600

aagggaatat aattaagcag gataccttta acatcatagg gaacgaccgt atgaagacta 3660

actatcacga taagctggca gctattgaga aggaccgcga ttcggcacgt aaggactgga 3720

agaagattaa caacatcaaa gagatgaaag aaggctatct ctctcaggtc gtacacgaga 3780

ttgccaagct cgtgatagag tacaacgcta ttgtggtttt cgaagatttg aactttggct 3840

ttaaaagggg caggttcaaa gtggaaaagc aggtgtatca aaagctggag aaaatgttga 3900

ttgagaagct caattatctg gtgttcaaag acaatgagtt tgacaagacg ggaggtgtgc 3960

tccgggcata ccagctcact gcacccttcg agactttcaa aaaaatgggg aagcagacag 4020

gaattattta ttacgtccct gctggcttca caagcaagat ctgccccgta acgggattcg 4080

tcaaccagct gtacccaaag tatgaatctg tctccaaaag tcaggaattc ttctcgaagt 4140

tcgacaagat atgctataat ctcgataagg gctactttga gttctctttc gattacaaga 4200

actttgggga taaagccgcg aagggtaaat ggactatagc gtcctttggg tcgcgcttga 4260

taaatttccg gaatagcgat aaaaatcaca actgggacac ccgcgaagtc tatcctacaa 4320

aagaacttga aaaactcctt aaggattaca gcatcgaata cgggcacggc gagtgtatca 4380

aagcggcgat ctgtggcgag tccgataaaa agttctttgc gaagctcaca tcagtcctga 4440

atacgatctt gcagatgcgg aactctaaaa ctgggacaga gctcgattac ctgatcagtc 4500

ctgtggccga cgttaacggg aacttctttg actcgcggca ggcgccaaag aacatgccac 4560

aggatgcgga tgccaacggg gcctatcata ttggcctcaa gggtctcatg ctcctgggaa 4620

gaattaagaa taatcaagaa ggcaaaaaac tcaatctcgt aattaagaat gaggaatact 4680

tcgagttcgt tcagaaccgt aacaataagc gtccggcagc gaccaagaag gccggacagg 4740

ctaaaaagaa gaagggaagt tacccttatg acgttcctga ttacgcgtat ccttatgacg 4800

tcccggacta tgcttaccct tacgatgttc cagattacgc ctaacccggg aattcggtac 4860

gctgaaatca ccagtctctc tctacaaatc tatctctctc tattttctcc ataaataatg 4920

tgtgagtagt ttcccgataa gggaaattag ggttcttata gggtttcgct catgtgttga 4980

gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc aataaaattt 5040

ctaattccta aaaccaaaat ccagtactaa aatccagatc tcctaaagtc cctatagatc 5100

tttgtcgtga atataaacca gacacgagac gactaaacct ggagcccaga cgccgttcga 5160

agctagaagt accgcttagg caggaggccg ttagggaaaa gatgctaagg cagggttggt 5220

tacgttgact cccccgtagg tttggtttaa atatgatgaa gtggacggaa ggaaggagga 5280

agacaaggaa ggataaggtt gcaggccctg tgcaaggtaa gaagatggaa atttgataga 5340

ggtacgctac tatacttata ctatacgcta agggaatgct tgtatttata ccctataccc 5400

cctaataacc ccttatcaat ttaagaaata atccgcataa gcccccgctt aaaaattggt 5460

atcagagcca tgaataggtc tatgaccaaa actcaagagg ataaaacctc accaaaatac 5520

gaaagagttc ttaactctaa agataaaaga tctttcaaga tcaaaa 5566

<210> 7

<211> 3921

<212> DNA

<213> Artificial sequence (AsCas 12 a)

<400> 7

atgacacagt ttgaagggtt cacaaacctt tatcaagtgt ccaaaacact tcgctttgag 60

cttataccac aaggaaaaac cctgaagcac atacaggagc aaggttttat cgaggaggac 120

aaggcccgca acgatcacta taaggaactc aaaccgatta tcgaccgcat atacaagacc 180

tacgctgacc agtgcctcca actggtccag ttggactggg agaacctgtc ggctgccata 240

gattcatatc gtaaggaaaa aactgaggag accagaaatg ccctcataga ggagcaggca 300

acatatagga acgccatcca cgactatttt attgggagaa cggacaacct cacggatgct 360

attaataagc gtcacgccga gatttacaaa ggcctgttta aggcggagtt gtttaatggc 420

aaggtcttga aacagctggg gacagtgact actactgagc atgaaaacgc ccttctccgc 480

tcttttgata agttcaccac ctatttctcg ggtttctacg agaatcgcaa gaacgtgttc 540

tccgcagaag acatttccac ggcaatcccc caccgcatcg ttcaggacaa ttttccgaaa 600

tttaaggaaa actgccacat tttcacccgg ctgataacag cagttcctag tctgcgtgaa 660

cattttgaga atgtgaagaa ggcgattggc atctttgttt caacttcaat tgaggaggtt 720

ttcagctttc ctttttacaa tcaactgctg acccagacgc agatagatct gtacaaccag 780

ctcctcggag ggatttcaag ggaagcgggc accgaaaaga tcaaaggctt gaacgaggtg 840

ctcaacctgg ctatccaaaa gaacgacgaa accgctcaca taattgccag cctcccacac 900

aggttcattc cgctgtttaa acagatcctg tctgacagaa acacactctc ttttattctg 960

gaggagttca aatccgacga ggaggttatt caatctttct gcaagtacaa aacactgctc 1020

cgcaacgaga acgtccttga gaccgcggag gcgctcttta atgaactcaa ttcgatagat 1080

cttacccaca tatttatttc tcacaagaag ctggagacga tctcgagcgc tctctgtgat 1140

cactgggaca cacttcggaa cgctctttat gagaggagga taagcgagct cacgggaaaa 1200

atcaccaaat ctgccaaaga gaaagtccag agatctctga aacatgagga tatcaacctc 1260

caggaaatta tatcggctgc gggaaaggag ctctcggaag cctttaagca gaaaacttct 1320

gagatcttga gtcatgctca tgctgccctc gatcaacctc ttcctaccac gttgaagaaa 1380

caggaggaga aagagatcct taagtctcag cttgattcat tgcttggttt gtaccatttg 1440

ctcgattggt tcgccgtaga tgagagcaat gaagtcgatc cagaattctc tgcgcggctg 1500

actggcatca agctggagat ggagccctcc ctctccttct ataacaaggc ccggaactat 1560

gcaactaaga agccatactc ggttgagaaa ttcaaactta acttccaaat gccaaccctc 1620

gcatccggct gggatgtgaa caaggagaag aacaatggag ctattctgtt tgttaagaat 1680

gggctttact atcttggcat tatgccaaaa caaaagggac gctacaaggc actctctttc 1740

gaaccgactg aaaaaacctc cgaaggattc gacaaaatgt attacgacta cttccctgac 1800

gcagcgaaaa tgatcccgaa gtgctcaact cagctgaagg cggtgaccgc gcatttccag 1860

acccatacga cccccattct cctttcgaat aatttcattg agcccctcga aattacaaag 1920

gagatctatg acctcaataa tcccgaaaaa gagccaaaga aatttcaaac ggcatatgca 1980

aagaaaacgg gcgaccaaaa gggttacagg gaggcgctct gtaagtggat agacttcact 2040

cgcgatttcc tgtctaagta taccaagacg acatccatag atctctcgtc gctcagacca 2100

tccagccagt ataaggatct gggggaatac tacgctgagc ttaatccgct gctctatcat 2160

atttcattcc aaagaattgc agagaaggag attatggatg ctgtggaaac ggggaaactc 2220

tacttgttcc agatttataa caaagatttc gccaagggtc atcatggtaa gccgaatctc 2280

cacacactgt attggacggg ccttttctcc cccgagaacc ttgccaaaac cagtatcaag 2340

cttaatgggc aggccgaact tttttatcgt cctaagagca gaatgaagag aatggctcat 2400

cgtttggggg aaaagatgtt gaacaagaaa ctgaaggatc aaaaaactcc tatcccggac 2460

acgctttatc aggagctgta tgattacgtc aatcacagat tgtcgcatga cctgtccgat 2520

gaggcgagag ccttgcttcc caacgtgatc acgaaggagg ttagccacga gataattaaa 2580

gacaggaggt tcacgtctga taaattcttc tttcatgtcc caataaccct taactatcag 2640

gcagccaatt ctcctagcaa attcaaccaa agggtgaacg catatcttaa ggaacatccg 2700

gagacgccca taatcgggat tgaccgcggc gagagaaatc tgatctatat cacggtaatc 2760

gacagtaccg ggaagatcct ggagcagcgt tcacttaata cgattcaaca attcgactac 2820

caaaaaaaac ttgacaatag agaaaaggaa cgggttgcag ccaggcaagc ctggtcggtg 2880

gtgggcacca taaaggatct taagcagggc tacctttcac aggttatcca cgagatagtg 2940

gatctgatga tccattatca agcagtcgtc gtgctcgaga atcttaattt cggattcaag 3000

agcaagcgga ctgggattgc tgaaaaggca gtgtaccaac aattcgagaa gatgctcatc 3060

gacaagctca attgcttggt cctgaaagac tatcccgcgg aaaaggtagg aggcgtcctc 3120

aatccctacc aactcacaga ccagtttact tcgtttgcaa aaatggggac ccagagcggc 3180

ttcctcttct acgtccctgc gccatacact tctaagattg acccgctcac cggctttgta 3240

gaccccttcg tgtggaagac tattaaaaat catgaatctc gcaagcactt cctggagggc 3300

tttgatttcc tccactatga cgtaaagacc ggggatttca tcctccattt caagatgaat 3360

agaaatctct ctttccaaag aggactccca ggcttcatgc ccgcctggga tatcgtattt 3420

gaaaagaacg aaacacaatt tgacgccaag ggcaccccct tcattgccgg aaagcggatt 3480

gttcctgtga ttgaaaacca tcgctttacc gggagataca gagaccttta tccggccaat 3540

gagctcattg cactcctcga ggagaaaggt attgtcttta gggatggctc gaacattctt 3600

ccgaagctcc ttgagaacga cgatagccat gcaattgata ctatggtcgc cttgattcgg 3660

tccgtattgc aaatgcgtaa ctcaaatgcc gcgacggggg aggactacat aaactctccc 3720

gttcgggacc tcaacggcgt ttgttttgat tcgcgcttcc aaaacccgga gtggcccatg 3780

gacgccgacg ccaacggtgc ctatcacata gctctgaaag gccagctgct tctcaatcac 3840

ctcaaggagt cgaaggacct gaagttgcaa aacgggataa gcaatcagga ttggctcgca 3900

tacatacaag agcttagaaa c 3921

<210> 8

<211> 3684

<212> DNA

<213> Artificial sequence (LbCas 12 a)

<400> 8

atgtctaagc tggagaagtt taccaactgc tactccttgt caaagacact cagattcaaa 60

gccatccctg tcggcaaaac acaggaaaat atcgataata agaggctctt ggtcgaggat 120

gaaaagcgtg ccgaggatta caagggtgtc aaaaagctgc tcgataggta ctatctgagc 180

ttcataaacg acgtcttgca ctccatcaag ctgaagaact tgaacaacta tatctctctc 240

tttaggaaga agacgagaac cgaaaaggaa aataaggagc tcgaaaactt ggaaattaat 300

ctgaggaagg agatagctaa ggcgttcaaa ggcaatgagg gatacaaatc cctgttcaag 360

aaggatataa tagagaccat ccttcctgag tttttggatg acaaggatga gattgcactc 420

gttaactcgt ttaatgggtt caccactgcg ttcaccggct tctttgataa tcgtgaaaac 480

atgttcagtg aagaagcgaa gtccacatcc atcgccttcc ggtgcataaa tgagaacctc 540

acacgctaca tctcgaacat ggatatattc gaaaaggtgg acgctatttt cgataaacac 600

gaggtgcagg agatcaagga gaagattttg aacagcgatt atgatgtgga agactttttc 660

gaaggagagt tcttcaactt cgtgctgacg caggagggta tcgatgttta caatgccatc 720

attggaggct tcgtcactga gagcggtgaa aagattaagg gtctgaacga gtatataaat 780

ctctacaatc aaaagacgaa gcagaagctg ccgaagttca aacctttgta caaacaagtg 840

ctctcggacc gcgagagcct ctccttttac ggagaagggt acacatcgga cgaggaggtt 900

ttggaagtct ttcgcaacac ccttaacaaa aactccgaga ttttttcctc gataaagaaa 960

cttgagaagt tgttcaagaa ttttgatgag tattcgtccg cgggcatctt cgtgaagaat 1020

ggtcctgcaa tatcgacgat cagcaaggac atatttggcg aatggaacgt cattcgggat 1080

aaatggaacg cagagtatga tgacatacat ctcaaaaaga aggctgtggt gactgaaaaa 1140

tacgaagatg atcggcggaa gtcgtttaag aagatcggtt cgttcagtct ggagcaactc 1200

caggagtatg ccgacgcaga tttgagtgtg gtcgaaaagc tcaaggaaat catcatccaa 1260

aaggtggatg aaatttacaa ggtgtacggt tcttcagaaa agctgttcga tgctgatttc 1320

gtgctcgaaa agagcctgaa gaagaatgat gctgtggttg cgatcatgaa ggaccttctc 1380

gatagcgtta agagctttga gaattatata aaggcatttt ttggagaggg caaggagact 1440

aatcgcgatg agagtttcta cggcgacttc gtgctggcat atgacattct tttgaaagtt 1500

gaccacattt atgatgccat ccgtaactat gtcacgcaga agccttacag taaagacaaa 1560

ttcaagttgt acttccagaa ccctcagttc atgggtggct gggacaagga caaagaaacg 1620

gattaccggg ccaccatcct tcgctatgga agtaagtact atctggcgat tatggataaa 1680

aaatacgcga aatgcctgca gaaaattgat aaagatgatg tcaatggcaa ctatgagaag 1740

attaactata aactgctccc tgggcccaat aagatgctcc cgaaggtatt cttcagtaag 1800

aagtggatgg cgtattataa cccttccgag gacatccaga aaatttacaa gaatggaacg 1860

ttcaagaagg gcgacatgtt taacctcaac gattgtcaca agttgatcga tttttttaaa 1920

gatagcatct cgcgttaccc caagtggagt aatgcgtatg actttaattt ctcggagacc 1980

gagaaatata aggatattgc gggtttctac cgtgaggtag aggagcaagg atataaggtt 2040

tccttcgaga gtgcttccaa gaaggaagtc gataagctgg tcgaggaggg caagctgtac 2100

atgttccaga tttacaacaa agatttctct gataagagcc acggtacgcc caacctgcat 2160

accatgtatt ttaagctcct cttcgacgag aacaaccacg ggcagatcag gttgtccggg 2220

ggcgcggagc tcttcatgag gagagcctct ctcaaaaagg aggagctggt ggttcaccca 2280

gctaattcgc ccatagccaa caagaatcct gacaatccca agaagacgac tacgttgagc 2340

tacgacgtat ataaagacaa gcggttctcc gaggaccaat acgaactcca tatccccatc 2400

gccataaata agtgcccaaa gaacattttt aagatcaaca ccgaagtgag agtgctcttg 2460

aagcatgacg acaatccgta tgttattggc atagacaggg gagagcggaa tctgctctac 2520

atagtcgttg tcgatggaaa gggcaatata gtcgaacagt actccttgaa cgagatcatc 2580

aacaatttca acggaatccg tatcaagacc gattaccact ccctcctgga caaaaaggag 2640

aaggagcggt tcgaggcgag gcaaaactgg acgagcattg agaacattaa ggagctcaaa 2700

gccggttaca tcagtcaggt cgtccacaag atctgcgaac tcgtagaaaa atatgacgct 2760

gtgatcgcct tggaggacct taactctggt tttaagaact caagagtaaa agttgagaag 2820

caggtttacc agaagtttga gaaaatgctc atagacaagc tgaattatat ggtagataaa 2880

aaatctaacc catgcgcgac tggcggagcg cttaagggat atcaaatcac taacaaattc 2940

gagtccttta aatcgatgtc cacccaaaat ggattcatct tttatatccc agcttggctt 3000

acgtcaaaga tcgatccaag cacgggattc gtgaaccttt tgaagaccaa gtatacgtcg 3060

attgcggact ccaaaaaatt catctcgtcc ttcgacagga tcatgtacgt gcccgaggag 3120

gatctcttcg aatttgcact ggattacaag aacttttctc gcaccgacgc cgattacatc 3180

aaaaaatgga agctctactc ttacggcaac cgtattcgga ttttccggaa tccgaaaaaa 3240

aataacgtgt tcgactggga ggaggtctgt ttgacctctg catataaaga gctctttaac 3300

aagtatggga tcaactatca gcaaggagat attagggcat tgctttgcga gcagtctgat 3360

aaagctttct actcaagttt catggcgctc atgtctctga tgttgcagat gaggaattcc 3420

atcacaggcc gcacagatgt ggacttcctc atatcgcctg tcaaaaacag cgatggcatc 3480

ttctacgact cacggaacta tgaagcgcag gagaatgcta ttcttcctaa aaatgcggac 3540

gcgaacggtg catacaacat tgccagaaaa gtactctggg caatcggtca attcaagaag 3600

gctgaggatg aaaagttgga taaggtcaag atcgctatct cgaataaaga atggttggag 3660

tatgctcaaa catcagttaa gcac 3684

<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> 24

<212> DNA

<213> Artificial sequence (gRNA 4)

<400> 12

cagtcgataa acccgccaaa cctg 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence (gRNA 5)

<400> 13

cacctaaaac atctcttgct tcaa 24

<210> 14

<211> 15222

<212> DNA

<213> Artificial sequence (PagPDS-AsCas 12 a)

<400> 14

aattcgagct cggtaccaaa aaaattgaag caagagatgt tttaggtgat ctacacttag 60

tagaaattac aggtttggcg ggtttatcga ctgatctaca cttagtagaa attagtttag 120

ctggacgcgg agaagaacat ctacacttag tagaaattaa atggcccatt gattcactgc 180

ctcatctaca cttagtagaa attatgcatt gaacttgagc tggcatagat ctacacttag 240

tagaaattac aatcactact tcgactctag ctgtatataa actcagcttc gttttcttat 300

ctaagcgatg tgggactttt gaagattgtt ttcaacttaa atgggcctat ataagaaata 360

ctattgttct ttcccatata aatgggcctg cttctcttct ttcagattcc caggggcctt 420

ttgaagatta tcttcatatc ttaagaatga agatgtttta ttcaatcaaa ttcttgaagg 480

ttcgatgcct aatcattcta atcctgggac aaactatgaa acaagataca aaaactccga 540

atggaaagtt aaaaagaaga aaacgaaagc tacggttcaa gaaaatgtaa gctgataaac 600

aaaaaaaaac tgtatgaacg aagaagaaga aaaaaagcta agaagaaatg atgtattgtg 660

cggaaggcaa gtcgagtttc cgttgttcaa cgaagcttgg atccgtaccc ctactccaaa 720

aatgtcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 780

atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca 840

gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggctatcatt 900

caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 960

gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga catctccact 1020

gacgtaaggg atgacgcaca atcccacccc tactccaaaa atgtcaaaga tacagtctca 1080

gaagaccaaa gggctattga gacttttcaa caaagggtaa tttcgggaaa cctcctcgga 1140

ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga aggtggctcc 1200

tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatgcctc tgccgacagt 1260

ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 1320

acgtcttcaa agcaagtgga ttgatgtgac atctccactg acgtaaggga tgacgcacaa 1380

tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg 1440

acagcccaag cttgtcgaca tggcccctaa gaagaagaga aaggtcggta ttcacggcgt 1500

tcctgcggcg atgacacagt ttgaagggtt cacaaacctt tatcaagtgt ccaaaacact 1560

tcgctttgag cttataccac aaggaaaaac cctgaagcac atacaggagc aaggttttat 1620

cgaggaggac aaggcccgca acgatcacta taaggaactc aaaccgatta tcgaccgcat 1680

atacaagacc tacgctgacc agtgcctcca actggtccag ttggactggg agaacctgtc 1740

ggctgccata gattcatatc gtaaggaaaa aactgaggag accagaaatg ccctcataga 1800

ggagcaggca acatatagga acgccatcca cgactatttt attgggagaa cggacaacct 1860

cacggatgct attaataagc gtcacgccga gatttacaaa ggcctgttta aggcggagtt 1920

gtttaatggc aaggtcttga aacagctggg gacagtgact actactgagc atgaaaacgc 1980

ccttctccgc tcttttgata agttcaccac ctatttctcg ggtttctacg agaatcgcaa 2040

gaacgtgttc tccgcagaag acatttccac ggcaatcccc caccgcatcg ttcaggacaa 2100

ttttccgaaa tttaaggaaa actgccacat tttcacccgg ctgataacag cagttcctag 2160

tctgcgtgaa cattttgaga atgtgaagaa ggcgattggc atctttgttt caacttcaat 2220

tgaggaggtt ttcagctttc ctttttacaa tcaactgctg acccagacgc agatagatct 2280

gtacaaccag ctcctcggag ggatttcaag ggaagcgggc accgaaaaga tcaaaggctt 2340

gaacgaggtg ctcaacctgg ctatccaaaa gaacgacgaa accgctcaca taattgccag 2400

cctcccacac aggttcattc cgctgtttaa acagatcctg tctgacagaa acacactctc 2460

ttttattctg gaggagttca aatccgacga ggaggttatt caatctttct gcaagtacaa 2520

aacactgctc cgcaacgaga acgtccttga gaccgcggag gcgctcttta atgaactcaa 2580

ttcgatagat cttacccaca tatttatttc tcacaagaag ctggagacga tctcgagcgc 2640

tctctgtgat cactgggaca cacttcggaa cgctctttat gagaggagga taagcgagct 2700

cacgggaaaa atcaccaaat ctgccaaaga gaaagtccag agatctctga aacatgagga 2760

tatcaacctc caggaaatta tatcggctgc gggaaaggag ctctcggaag cctttaagca 2820

gaaaacttct gagatcttga gtcatgctca tgctgccctc gatcaacctc ttcctaccac 2880

gttgaagaaa caggaggaga aagagatcct taagtctcag cttgattcat tgcttggttt 2940

gtaccatttg ctcgattggt tcgccgtaga tgagagcaat gaagtcgatc cagaattctc 3000

tgcgcggctg actggcatca agctggagat ggagccctcc ctctccttct ataacaaggc 3060

ccggaactat gcaactaaga agccatactc ggttgagaaa ttcaaactta acttccaaat 3120

gccaaccctc gcatccggct gggatgtgaa caaggagaag aacaatggag ctattctgtt 3180

tgttaagaat gggctttact atcttggcat tatgccaaaa caaaagggac gctacaaggc 3240

actctctttc gaaccgactg aaaaaacctc cgaaggattc gacaaaatgt attacgacta 3300

cttccctgac gcagcgaaaa tgatcccgaa gtgctcaact cagctgaagg cggtgaccgc 3360

gcatttccag acccatacga cccccattct cctttcgaat aatttcattg agcccctcga 3420

aattacaaag gagatctatg acctcaataa tcccgaaaaa gagccaaaga aatttcaaac 3480

ggcatatgca aagaaaacgg gcgaccaaaa gggttacagg gaggcgctct gtaagtggat 3540

agacttcact cgcgatttcc tgtctaagta taccaagacg acatccatag atctctcgtc 3600

gctcagacca tccagccagt ataaggatct gggggaatac tacgctgagc ttaatccgct 3660

gctctatcat atttcattcc aaagaattgc agagaaggag attatggatg ctgtggaaac 3720

ggggaaactc tacttgttcc agatttataa caaagatttc gccaagggtc atcatggtaa 3780

gccgaatctc cacacactgt attggacggg ccttttctcc cccgagaacc ttgccaaaac 3840

cagtatcaag cttaatgggc aggccgaact tttttatcgt cctaagagca gaatgaagag 3900

aatggctcat cgtttggggg aaaagatgtt gaacaagaaa ctgaaggatc aaaaaactcc 3960

tatcccggac acgctttatc aggagctgta tgattacgtc aatcacagat tgtcgcatga 4020

cctgtccgat gaggcgagag ccttgcttcc caacgtgatc acgaaggagg ttagccacga 4080

gataattaaa gacaggaggt tcacgtctga taaattcttc tttcatgtcc caataaccct 4140

taactatcag gcagccaatt ctcctagcaa attcaaccaa agggtgaacg catatcttaa 4200

ggaacatccg gagacgccca taatcgggat tgaccgcggc gagagaaatc tgatctatat 4260

cacggtaatc gacagtaccg ggaagatcct ggagcagcgt tcacttaata cgattcaaca 4320

attcgactac caaaaaaaac ttgacaatag agaaaaggaa cgggttgcag ccaggcaagc 4380

ctggtcggtg gtgggcacca taaaggatct taagcagggc tacctttcac aggttatcca 4440

cgagatagtg gatctgatga tccattatca agcagtcgtc gtgctcgaga atcttaattt 4500

cggattcaag agcaagcgga ctgggattgc tgaaaaggca gtgtaccaac aattcgagaa 4560

gatgctcatc gacaagctca attgcttggt cctgaaagac tatcccgcgg aaaaggtagg 4620

aggcgtcctc aatccctacc aactcacaga ccagtttact tcgtttgcaa aaatggggac 4680

ccagagcggc ttcctcttct acgtccctgc gccatacact tctaagattg acccgctcac 4740

cggctttgta gaccccttcg tgtggaagac tattaaaaat catgaatctc gcaagcactt 4800

cctggagggc tttgatttcc tccactatga cgtaaagacc ggggatttca tcctccattt 4860

caagatgaat agaaatctct ctttccaaag aggactccca ggcttcatgc ccgcctggga 4920

tatcgtattt gaaaagaacg aaacacaatt tgacgccaag ggcaccccct tcattgccgg 4980

aaagcggatt gttcctgtga ttgaaaacca tcgctttacc gggagataca gagaccttta 5040

tccggccaat gagctcattg cactcctcga ggagaaaggt attgtcttta gggatggctc 5100

gaacattctt ccgaagctcc ttgagaacga cgatagccat gcaattgata ctatggtcgc 5160

cttgattcgg tccgtattgc aaatgcgtaa ctcaaatgcc gcgacggggg aggactacat 5220

aaactctccc gttcgggacc tcaacggcgt ttgttttgat tcgcgcttcc aaaacccgga 5280

gtggcccatg gacgccgacg ccaacggtgc ctatcacata gctctgaaag gccagctgct 5340

tctcaatcac ctcaaggagt cgaaggacct gaagttgcaa aacgggataa gcaatcagga 5400

ttggctcgca tacatacaag agcttagaaa caagcgtccg gcagcgacca agaaggccgg 5460

acaggctaaa aagaagaagg gaagttaccc ttatgacgtt cctgattacg cgtatcctta 5520

tgacgtcccg gactatgctt acccttacga tgttccagat tacgcctaac ccgggaattc 5580

ggtacgctga aatcaccagt ctctctctac aaatctatct ctctctattt tctccataaa 5640

taatgtgtga gtagtttccc gataagggaa attagggttc ttatagggtt tcgctcatgt 5700

gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 5760

aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatctccta aagtccctat 5820

agatctttgt cgtgaatata aaccagacac gagacgacta aacctggagc ccagacgccg 5880

ttcgaagcta gaagtaccgc ttaggcagga ggccgttagg gaaaagatgc taaggcaggg 5940

ttggttacgt tgactccccc gtaggtttgg tttaaatatg atgaagtgga cggaaggaag 6000

gaggaagaca aggaaggata aggttgcagg ccctgtgcaa ggtaagaaga tggaaatttg 6060

atagaggtac gctactatac ttatactata cgctaaggga atgcttgtat ttatacccta 6120

taccccctaa taacccctta tcaatttaag aaataatccg cataagcccc cgcttaaaaa 6180

ttggtatcag agccatgaat aggtctatga ccaaaactca agaggataaa acctcaccaa 6240

aatacgaaag agttcttaac tctaaagata aaagatcttt caagatcaaa actagagtcg 6300

acctgcaggc atgcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6360

cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6420

agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc 6480

tagagcagct tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg 6540

tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga 6600

tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac 6660

agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg 6720

gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta 6780

cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca 6840

taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg 6900

ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg 6960

ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc 7020

gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag 7080

tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca 7140

tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc 7200

cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa 7260

tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc 7320

cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag 7380

gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg 7440

cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg 7500

aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac 7560

aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga 7620

ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg 7680

gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt 7740

ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag 7800

cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg 7860

gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc 7920

gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc 7980

gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt 8040

gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc 8100

gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc 8160

gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg 8220

gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc 8280

gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg 8340

tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc 8400

gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag 8460

gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga 8520

gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa 8580

cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg 8640

aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc 8700

tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga 8760

attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg 8820

gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc 8880

cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc 8940

atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 9000

cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 9060

agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 9120

gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 9180

cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 9240

tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 9300

cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 9360

ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 9420

gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 9480

gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 9540

tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 9600

ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc 9660

tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 9720

cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 9780

cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 9840

cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 9900

ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 9960

caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 10020

aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 10080

tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 10140

cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 10200

cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 10260

gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 10320

cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 10380

cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 10440

gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 10500

acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 10560

cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 10620

gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 10680

agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 10740

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 10800

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 10860

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 10920

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 10980

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 11040

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 11100

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 11160

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 11220

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 11280

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 11340

gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 11400

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 11460

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 11520

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 11580

tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt 11640

ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc 11700

gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc 11760

gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc 11820

tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc 11880

atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc 11940

ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt 12000

atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc 12060

gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc 12120

ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac 12180

aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt 12240

ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt 12300

atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag 12360

ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta 12420

cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc 12480

cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg 12540

gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg 12600

ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg 12660

gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta 12720

caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt 12780

ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg 12840

taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa 12900

ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt 12960

agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat 13020

gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac 13080

acattattat ggagaaactc gagcttgtcg atcgacagat ccggtcggca tctactctat 13140

ttctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca 13200

cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg 13260

gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg 13320

ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagt 13380

cgtggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata 13440

caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac 13500

atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg 13560

gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc 13620

agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag 13680

tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg 13740

attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc 13800

gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct 13860

tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc gctaaactcc 13920

ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa 13980

cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct 14040

acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg 14100

ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc 14160

atatctcatt gccccccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga 14220

ctggtgattt cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct 14280

tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc 14340

acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg 14400

ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg 14460

caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag 14520

atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca 14580

atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg 14640

tgctccacca tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct 14700

ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca 14760

tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt 14820

tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc 14880

gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg 14940

atattcttgg agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa 15000

tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 15060

ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 15120

aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 15180

gataacaatt tcacacagga aacagctatg accatgatta cg 15222

<210> 15

<211> 14985

<212> DNA

<213> Artificial sequence (PagPDS-LbCas 12 a)

<400> 15

aattcgagct cggtaccaaa aaaattgaag caagagatgt tttaggtgat ctacacttag 60

tagaaattac aggtttggcg ggtttatcga ctgatctaca cttagtagaa attagtttag 120

ctggacgcgg agaagaacat ctacacttag tagaaattaa atggcccatt gattcactgc 180

ctcatctaca cttagtagaa attatgcatt gaacttgagc tggcatagat ctacacttag 240

tagaaattac aatcactact tcgactctag ctgtatataa actcagcttc gttttcttat 300

ctaagcgatg tgggactttt gaagattgtt ttcaacttaa atgggcctat ataagaaata 360

ctattgttct ttcccatata aatgggcctg cttctcttct ttcagattcc caggggcctt 420

ttgaagatta tcttcatatc ttaagaatga agatgtttta ttcaatcaaa ttcttgaagg 480

ttcgatgcct aatcattcta atcctgggac aaactatgaa acaagataca aaaactccga 540

atggaaagtt aaaaagaaga aaacgaaagc tacggttcaa gaaaatgtaa gctgataaac 600

aaaaaaaaac tgtatgaacg aagaagaaga aaaaaagcta agaagaaatg atgtattgtg 660

cggaaggcaa gtcgagtttc cgttgttcaa cgaagcttgg atccgtaccc ctactccaaa 720

aatgtcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 780

atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca 840

gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggctatcatt 900

caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 960

gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga catctccact 1020

gacgtaaggg atgacgcaca atcccacccc tactccaaaa atgtcaaaga tacagtctca 1080

gaagaccaaa gggctattga gacttttcaa caaagggtaa tttcgggaaa cctcctcgga 1140

ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga aggtggctcc 1200

tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatgcctc tgccgacagt 1260

ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 1320

acgtcttcaa agcaagtgga ttgatgtgac atctccactg acgtaaggga tgacgcacaa 1380

tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg 1440

acagcccaag cttgtcgaca tggcccctaa gaagaagaga aaggtcggta ttcacggcgt 1500

tcctgcggcg atgtctaagc tggagaagtt taccaactgc tactccttgt caaagacact 1560

cagattcaaa gccatccctg tcggcaaaac acaggaaaat atcgataata agaggctctt 1620

ggtcgaggat gaaaagcgtg ccgaggatta caagggtgtc aaaaagctgc tcgataggta 1680

ctatctgagc ttcataaacg acgtcttgca ctccatcaag ctgaagaact tgaacaacta 1740

tatctctctc tttaggaaga agacgagaac cgaaaaggaa aataaggagc tcgaaaactt 1800

ggaaattaat ctgaggaagg agatagctaa ggcgttcaaa ggcaatgagg gatacaaatc 1860

cctgttcaag aaggatataa tagagaccat ccttcctgag tttttggatg acaaggatga 1920

gattgcactc gttaactcgt ttaatgggtt caccactgcg ttcaccggct tctttgataa 1980

tcgtgaaaac atgttcagtg aagaagcgaa gtccacatcc atcgccttcc ggtgcataaa 2040

tgagaacctc acacgctaca tctcgaacat ggatatattc gaaaaggtgg acgctatttt 2100

cgataaacac gaggtgcagg agatcaagga gaagattttg aacagcgatt atgatgtgga 2160

agactttttc gaaggagagt tcttcaactt cgtgctgacg caggagggta tcgatgttta 2220

caatgccatc attggaggct tcgtcactga gagcggtgaa aagattaagg gtctgaacga 2280

gtatataaat ctctacaatc aaaagacgaa gcagaagctg ccgaagttca aacctttgta 2340

caaacaagtg ctctcggacc gcgagagcct ctccttttac ggagaagggt acacatcgga 2400

cgaggaggtt ttggaagtct ttcgcaacac ccttaacaaa aactccgaga ttttttcctc 2460

gataaagaaa cttgagaagt tgttcaagaa ttttgatgag tattcgtccg cgggcatctt 2520

cgtgaagaat ggtcctgcaa tatcgacgat cagcaaggac atatttggcg aatggaacgt 2580

cattcgggat aaatggaacg cagagtatga tgacatacat ctcaaaaaga aggctgtggt 2640

gactgaaaaa tacgaagatg atcggcggaa gtcgtttaag aagatcggtt cgttcagtct 2700

ggagcaactc caggagtatg ccgacgcaga tttgagtgtg gtcgaaaagc tcaaggaaat 2760

catcatccaa aaggtggatg aaatttacaa ggtgtacggt tcttcagaaa agctgttcga 2820

tgctgatttc gtgctcgaaa agagcctgaa gaagaatgat gctgtggttg cgatcatgaa 2880

ggaccttctc gatagcgtta agagctttga gaattatata aaggcatttt ttggagaggg 2940

caaggagact aatcgcgatg agagtttcta cggcgacttc gtgctggcat atgacattct 3000

tttgaaagtt gaccacattt atgatgccat ccgtaactat gtcacgcaga agccttacag 3060

taaagacaaa ttcaagttgt acttccagaa ccctcagttc atgggtggct gggacaagga 3120

caaagaaacg gattaccggg ccaccatcct tcgctatgga agtaagtact atctggcgat 3180

tatggataaa aaatacgcga aatgcctgca gaaaattgat aaagatgatg tcaatggcaa 3240

ctatgagaag attaactata aactgctccc tgggcccaat aagatgctcc cgaaggtatt 3300

cttcagtaag aagtggatgg cgtattataa cccttccgag gacatccaga aaatttacaa 3360

gaatggaacg ttcaagaagg gcgacatgtt taacctcaac gattgtcaca agttgatcga 3420

tttttttaaa gatagcatct cgcgttaccc caagtggagt aatgcgtatg actttaattt 3480

ctcggagacc gagaaatata aggatattgc gggtttctac cgtgaggtag aggagcaagg 3540

atataaggtt tccttcgaga gtgcttccaa gaaggaagtc gataagctgg tcgaggaggg 3600

caagctgtac atgttccaga tttacaacaa agatttctct gataagagcc acggtacgcc 3660

caacctgcat accatgtatt ttaagctcct cttcgacgag aacaaccacg ggcagatcag 3720

gttgtccggg ggcgcggagc tcttcatgag gagagcctct ctcaaaaagg aggagctggt 3780

ggttcaccca gctaattcgc ccatagccaa caagaatcct gacaatccca agaagacgac 3840

tacgttgagc tacgacgtat ataaagacaa gcggttctcc gaggaccaat acgaactcca 3900

tatccccatc gccataaata agtgcccaaa gaacattttt aagatcaaca ccgaagtgag 3960

agtgctcttg aagcatgacg acaatccgta tgttattggc atagacaggg gagagcggaa 4020

tctgctctac atagtcgttg tcgatggaaa gggcaatata gtcgaacagt actccttgaa 4080

cgagatcatc aacaatttca acggaatccg tatcaagacc gattaccact ccctcctgga 4140

caaaaaggag aaggagcggt tcgaggcgag gcaaaactgg acgagcattg agaacattaa 4200

ggagctcaaa gccggttaca tcagtcaggt cgtccacaag atctgcgaac tcgtagaaaa 4260

atatgacgct gtgatcgcct tggaggacct taactctggt tttaagaact caagagtaaa 4320

agttgagaag caggtttacc agaagtttga gaaaatgctc atagacaagc tgaattatat 4380

ggtagataaa aaatctaacc catgcgcgac tggcggagcg cttaagggat atcaaatcac 4440

taacaaattc gagtccttta aatcgatgtc cacccaaaat ggattcatct tttatatccc 4500

agcttggctt acgtcaaaga tcgatccaag cacgggattc gtgaaccttt tgaagaccaa 4560

gtatacgtcg attgcggact ccaaaaaatt catctcgtcc ttcgacagga tcatgtacgt 4620

gcccgaggag gatctcttcg aatttgcact ggattacaag aacttttctc gcaccgacgc 4680

cgattacatc aaaaaatgga agctctactc ttacggcaac cgtattcgga ttttccggaa 4740

tccgaaaaaa aataacgtgt tcgactggga ggaggtctgt ttgacctctg catataaaga 4800

gctctttaac aagtatggga tcaactatca gcaaggagat attagggcat tgctttgcga 4860

gcagtctgat aaagctttct actcaagttt catggcgctc atgtctctga tgttgcagat 4920

gaggaattcc atcacaggcc gcacagatgt ggacttcctc atatcgcctg tcaaaaacag 4980

cgatggcatc ttctacgact cacggaacta tgaagcgcag gagaatgcta ttcttcctaa 5040

aaatgcggac gcgaacggtg catacaacat tgccagaaaa gtactctggg caatcggtca 5100

attcaagaag gctgaggatg aaaagttgga taaggtcaag atcgctatct cgaataaaga 5160

atggttggag tatgctcaaa catcagttaa gcacaagcgt ccggcagcga ccaagaaggc 5220

cggacaggct aaaaagaaga agggaagtta cccttatgac gttcctgatt acgcgtatcc 5280

ttatgacgtc ccggactatg cttaccctta cgatgttcca gattacgcct aacccgggaa 5340

ttcggtacgc tgaaatcacc agtctctctc tacaaatcta tctctctcta ttttctccat 5400

aaataatgtg tgagtagttt cccgataagg gaaattaggg ttcttatagg gtttcgctca 5460

tgtgttgagc atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa 5520

taaaatttct aattcctaaa accaaaatcc agtactaaaa tccagatctc ctaaagtccc 5580

tatagatctt tgtcgtgaat ataaaccaga cacgagacga ctaaacctgg agcccagacg 5640

ccgttcgaag ctagaagtac cgcttaggca ggaggccgtt agggaaaaga tgctaaggca 5700

gggttggtta cgttgactcc cccgtaggtt tggtttaaat atgatgaagt ggacggaagg 5760

aaggaggaag acaaggaagg ataaggttgc aggccctgtg caaggtaaga agatggaaat 5820

ttgatagagg tacgctacta tacttatact atacgctaag ggaatgcttg tatttatacc 5880

ctataccccc taataacccc ttatcaattt aagaaataat ccgcataagc ccccgcttaa 5940

aaattggtat cagagccatg aataggtcta tgaccaaaac tcaagaggat aaaacctcac 6000

caaaatacga aagagttctt aactctaaag ataaaagatc tttcaagatc aaaactagag 6060

tcgacctgca ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa 6120

aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 6180

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 6240

tgctagagca gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca 6300

gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac 6360

ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac 6420

cacagggttc ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag 6480

tcggcttctg acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag 6540

ttacgcgaca ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc 6600

gcataaagta gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg 6660

ccgctggcct gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac 6720

gggccgaact gcacgcggcc ggctgcacca agctgttttc cgagaagatc accggcacca 6780

ggcgcgaccg cccggagctg gccaggatgc ttgaccacct acgccctggc gacgttgtga 6840

cagtgaccag gctagaccgc ctggcccgca gcacccgcga cctactggac attgccgagc 6900

gcatccagga ggccggcgcg ggcctgcgta gcctggcaga gccgtgggcc gacaccacca 6960

cgccggccgg ccgcatggtg ttgaccgtgt tcgccggcat tgccgagttc gagcgttccc 7020

taatcatcga ccgcacccgg agcgggcgcg aggccgccaa ggcccgaggc gtgaagtttg 7080

gcccccgccc taccctcacc ccggcacaga tcgcgcacgc ccgcgagctg atcgaccagg 7140

aaggccgcac cgtgaaagag gcggctgcac tgcttggcgt gcatcgctcg accctgtacc 7200

gcgcacttga gcgcagcgag gaagtgacgc ccaccgaggc caggcggcgc ggtgccttcc 7260

gtgaggacgc attgaccgag gccgacgccc tggcggccgc cgagaatgaa cgccaagagg 7320

aacaagcatg aaaccgcacc aggacggcca ggacgaaccg tttttcatta ccgaagagat 7380

cgaggcggag atgatcgcgg ccgggtacgt gttcgagccg cccgcgcacg tctcaaccgt 7440

gcggctgcat gaaatcctgg ccggtttgtc tgatgccaag ctggcggcct ggccggccag 7500

cttggccgct gaagaaaccg agcgccgccg tctaaaaagg tgatgtgtat ttgagtaaaa 7560

cagcttgcgt catgcggtcg ctgcgtatat gatgcgatga gtaaataaac aaatacgcaa 7620

ggggaacgca tgaaggttat cgctgtactt aaccagaaag gcgggtcagg caagacgacc 7680

atcgcaaccc atctagcccg cgccctgcaa ctcgccgggg ccgatgttct gttagtcgat 7740

tccgatcccc agggcagtgc ccgcgattgg gcggccgtgc gggaagatca accgctaacc 7800

gttgtcggca tcgaccgccc gacgattgac cgcgacgtga aggccatcgg ccggcgcgac 7860

ttcgtagtga tcgacggagc gccccaggcg gcggacttgg ctgtgtccgc gatcaaggca 7920

gccgacttcg tgctgattcc ggtgcagcca agcccttacg acatatgggc caccgccgac 7980

ctggtggagc tggttaagca gcgcattgag gtcacggatg gaaggctaca agcggccttt 8040

gtcgtgtcgc gggcgatcaa aggcacgcgc atcggcggtg aggttgccga ggcgctggcc 8100

gggtacgagc tgcccattct tgagtcccgt atcacgcagc gcgtgagcta cccaggcact 8160

gccgccgccg gcacaaccgt tcttgaatca gaacccgagg gcgacgctgc ccgcgaggtc 8220

caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag ttaatgaggt aaagagaaaa 8280

tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag cgcacgcagc agcaaggctg 8340

caacgttggc cagcctggca gacacgccag ccatgaagcg ggtcaacttt cagttgccgg 8400

cggaggatca caccaagctg aagatgtacg cggtacgcca aggcaagacc attaccgagc 8460

tgctatctga atacatcgcg cagctaccag agtaaatgag caaatgaata aatgagtaga 8520

tgaattttag cggctaaagg aggcggcatg gaaaatcaag aacaaccagg caccgacgcc 8580

gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag gcgtaagcgg ctgggttgtc 8640

tgccggccct gcaatggcac tggaaccccc aagcccgagg aatcggcgtg acggtcgcaa 8700

accatccggc ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa 8760

ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg 8820

gcaagcggcc gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc 8880

gtcgattagg aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta 8940

tgacgtgggc acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa 9000

gcgtgaccga cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt 9060

ttccgcaggg ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt 9120

ttcccatcta accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg 9180

cgtgttccgt ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa 9240

gcagaaagac gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca 9300

gcgtacgaag aaggccaaga acggccgcct ggtgacggta tccgagggtg aagccttgat 9360

tagccgctac aagatcgtaa agagcgaaac cgggcggccg gagtacatcg agatcgagct 9420

agctgattgg atgtaccgcg agatcacaga aggcaagaac ccggacgtgc tgacggttca 9480

ccccgattac tttttgatcg atcccggcat cggccgtttt ctctaccgcc tggcacgccg 9540

cgccgcaggc aaggcagaag ccagatggtt gttcaagacg atctacgaac gcagtggcag 9600

cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct 9660

gccggagtac gatttgaagg aggaggcggg gcaggctggc ccgatcctag tcatgcgcta 9720

ccgcaacctg atcgagggcg aagcatccgc cggttcctaa tgtacggagc agatgctagg 9780

gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta 9840

cattgggaac ccaaagccgt acattgggaa ccggaacccg tacattggga acccaaagcc 9900

gtacattggg aaccggtcac acatgtaagt gactgatata aaagagaaaa aaggcgattt 9960

ttccgcctaa aactctttaa aacttattaa aactcttaaa acccgcctgg cctgtgcata 10020

actgtctggc cagcgcacag ccgaagagct gcaaaaagcg cctacccttc ggtcgctgcg 10080

ctccctacgc cccgccgctt cgcgtcggcc tatcgcggcc gctggccgct caaaaatggc 10140

tggcctacgg ccaggcaatc taccagggcg cggacaagcc gcgccgtcgc cactcgaccg 10200

ccggcgccca catcaaggca ccctgcctcg cgcgtttcgg tgatgacggt gaaaacctct 10260

gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 10320

aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt 10380

cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact 10440

gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 10500

caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 10560

agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 10620

aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 10680

gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 10740

tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 10800

cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 10860

ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 10920

cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 10980

atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 11040

agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 11100

gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 11160

gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 11220

tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 11280

agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 11340

gattttggtc atgcattcta ggtactaaaa caattcatcc agtaaaatat aatattttat 11400

tttctcccaa tcaggcttga tccccagtaa gtcaaaaaat agctcgacat actgttcttc 11460

cccgatatcc tccctgatcg accggacgca gaaggcaatg tcataccact tgtccgccct 11520

gccgcttctc ccaagatcaa taaagccact tactttgcca tctttcacaa agatgttgct 11580

gtctcccagg tcgccgtggg aaaagacaag ttcctcttcg ggcttttccg tctttaaaaa 11640

atcatacagc tcgcgcggat ctttaaatgg agtgtcttct tcccagtttt cgcaatccac 11700

atcggccaga tcgttattca gtaagtaatc caattcggct aagcggctgt ctaagctatt 11760

cgtataggga caatccgata tgtcgatgga gtgaaagagc ctgatgcact ccgcatacag 11820

ctcgataatc ttttcagggc tttgttcatc ttcatactct tccgagcaaa ggacgccatc 11880

ggcctcactc atgagcagat tgctccagcc atcatgccgt tcaaagtgca ggacctttgg 11940

aacaggcagc tttccttcca gccatagcat catgtccttt tcccgttcca catcataggt 12000

ggtcccttta taccggctgt ccgtcatttt taaatatagg ttttcatttt ctcccaccag 12060

cttatatacc ttagcaggag acattccttc cgtatctttt acgcagcggt atttttcgat 12120

cagttttttc aattccggtg atattctcat tttagccatt tattatttcc ttcctctttt 12180

ctacagtatt taaagatacc ccaagaagct aattataaca agacgaactc caattcactg 12240

ttccttgcat tctaaaacct taaataccag aaaacagctt tttcaaagtt gttttcaaag 12300

ttggcgtata acatagtatc gacggagccg attttgaaac cgcggtgatc acaggcagca 12360

acgctctgtc atcgttacaa tcaacatgct accctccgcg agatcatccg tgtttcaaac 12420

ccggcagctt agttgccgtt cttccgaata gcatcggtaa catgagcaaa gtctgccgcc 12480

ttacaacggc tctcccgctg acgccgtccc ggactgatgg gctgcctgta tcgagtggtg 12540

attttgtgcc gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg 12600

gtgtaaacaa attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact 12660

gaattaacgc cgaattaatt cgggggatct ggattttagt actggatttt ggttttagga 12720

attagaaatt ttattgatag aagtatttta caaatacaaa tacatactaa gggtttctta 12780

tatgctcaac acatgagcga aaccctatag gaaccctaat tcccttatct gggaactact 12840

cacacattat tatggagaaa ctcgagcttg tcgatcgaca gatccggtcg gcatctactc 12900

tatttctttg ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct 12960

acacagccat cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc 13020

ccggctccgg atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa 13080

ttgccgtcaa ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg 13140

agtcgtggcg atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc 13200

atacaagcca accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg 13260

aacatcgcct cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg 13320

ttggagccga aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc 13380

atcagctcat cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc 13440

cagtgataca catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga 13500

ccgattcctt gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg 13560

atcgcatcca tagcctccgc gaccggttgt agaacagcgg gcagttcggt ttcaggcagg 13620

tcttgcaacg tgacaccctg tgcacggcgg gagatgcaat aggtcaggct ctcgctaaac 13680

tccccaatgt caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca 13740

taacgatctt tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct 13800

cctacatcga agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag 13860

acgctgtcga acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt 13920

ttcatatctc attgcccccc gggatctgcg aaagctcgag agagatagat ttgtagagag 13980

agactggtga tttcagcgtg tcctctccaa atgaaatgaa cttccttata tagaggaagg 14040

tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt cagtggagat atcacatcaa 14100

tccacttgct ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt 14160

gggggtccat ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta 14220

tcgcaatgat ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga 14280

cagatagctg ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc 14340

tcaatagccc tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg 14400

tcgtgctcca ccatgttatc acatcaatcc acttgctttg aagacgtggt tggaacgtct 14460

tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact gtcggcagag 14520

gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt gccaccttcc 14580

ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc gaggaggttt 14640

cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga gactgtatct 14700

ttgatattct tggagtagac gagagtgtcg tgctccacca tgttggcaag ctgctctagc 14760

caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 14820

ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 14880

attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 14940

gcggataaca atttcacaca ggaaacagct atgaccatga ttacg 14985

<210> 16

<211> 15201

<212> DNA

<213> Artificial sequence (PagPDS-FnCas 12 a)

<400> 16

aattcgagct cggtaccaaa aaaattgaag caagagatgt tttaggtgat ctacacttag 60

tagaaattac aggtttggcg ggtttatcga ctgatctaca cttagtagaa attagtttag 120

ctggacgcgg agaagaacat ctacacttag tagaaattaa atggcccatt gattcactgc 180

ctcatctaca cttagtagaa attatgcatt gaacttgagc tggcatagat ctacacttag 240

tagaaattac aatcactact tcgactctag ctgtatataa actcagcttc gttttcttat 300

ctaagcgatg tgggactttt gaagattgtt ttcaacttaa atgggcctat ataagaaata 360

ctattgttct ttcccatata aatgggcctg cttctcttct ttcagattcc caggggcctt 420

ttgaagatta tcttcatatc ttaagaatga agatgtttta ttcaatcaaa ttcttgaagg 480

ttcgatgcct aatcattcta atcctgggac aaactatgaa acaagataca aaaactccga 540

atggaaagtt aaaaagaaga aaacgaaagc tacggttcaa gaaaatgtaa gctgataaac 600

aaaaaaaaac tgtatgaacg aagaagaaga aaaaaagcta agaagaaatg atgtattgtg 660

cggaaggcaa gtcgagtttc cgttgttcaa cgaagcttgg atccgtaccc ctactccaaa 720

aatgtcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaagggta 780

atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca 840

gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggctatcatt 900

caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 960

gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga catctccact 1020

gacgtaaggg atgacgcaca atcccacccc tactccaaaa atgtcaaaga tacagtctca 1080

gaagaccaaa gggctattga gacttttcaa caaagggtaa tttcgggaaa cctcctcgga 1140

ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga aggtggctcc 1200

tacaaatgcc atcattgcga taaaggaaag gctatcattc aagatgcctc tgccgacagt 1260

ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 1320

acgtcttcaa agcaagtgga ttgatgtgac atctccactg acgtaaggga tgacgcacaa 1380

tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg 1440

acagcccaag cttgtcgaca tggcccctaa gaagaagaga aaggtcggta ttcacggcgt 1500

tcctgcggcg atgtccatat atcaagagtt tgtgaataag tattcactga gtaagacact 1560

caggttcgag ctcatccctc agggcaaaac ccttgagaac atcaaggctc ggggcctcat 1620

actggatgac gaaaagcgcg ccaaagacta caagaaggcg aagcagatta tcgacaagta 1680

tcaccagttc tttattgagg agatcctctc ttcagtctgc atctctgagg atctcctgca 1740

aaactatagt gatgtctact tcaagctcaa gaagagcgat gatgataacc tccagaaaga 1800

tttcaagtca gccaaagaca cgattaaaaa acagatctcc gaatacatca aggattccga 1860

gaagtttaaa aatctgttca accaaaatct tattgacgcg aaaaagggcc aggaatcaga 1920

cttgatcctg tggctcaagc aatccaagga caatggcatc gagctgttta aagccaattc 1980

cgatattacc gatattgatg aagccttgga aatcataaag tcttttaagg gttggactac 2040

gtacttcaag gggtttcacg agaatcgcaa gaacgtttat tcaagtaatg acatacccac 2100

atctattatc tacagaattg tggatgacaa tctgcccaaa ttcttggaaa ataaagccaa 2160

gtatgaatcg cttaaggaca aggctccgga ggccatcaac tatgaacaaa taaagaagga 2220

tctggctgaa gagctgacct ttgatataga ttacaagacc tcggaagtga atcagcgggt 2280

gttcagcctc gatgaggtat ttgaaatagc aaactttaac aactacctca accaatccgg 2340

cattaccaaa tttaacacta ttattggcgg taaatttgtg aatggggaga acacgaagag 2400

gaaaggcatt aacgaatata tcaaccttta cagccagcag atcaatgata agaccctcaa 2460

gaagtacaaa atgagtgtcc tcttcaagca gatcctcagt gacaccgaga gcaaaagttt 2520

cgtaatagat aagcttgagg atgattccga cgtagtgact acaatgcaga gtttttatga 2580

acagatagct gcgttcaaga ccgtggaaga aaaatcgata aaagagacac tcagccttct 2640

gttcgacgac ctgaaggcgc aaaaacttga tctcagcaag atctatttta agaacgataa 2700

gtccctgaca gatctgtcgc agcaggtgtt tgatgactac agcgttatcg gcaccgcagt 2760

gctggagtat ataacccaac agatcgcacc gaaaaacctc gacaatccaa gcaagaagga 2820

acaagaactt atcgccaaga agacagagaa ggccaaatat ttgagcctcg agacgattaa 2880

gttggcgctg gaagagttca acaagcatcg ggacattgac aagcagtgca ggttcgagga 2940

gattttggcg aacttcgccg cgatcccgat gattttcgac gagatcgctc agaacaaaga 3000

caacctcgcc caaatctcga tcaagtacca gaatcagggg aagaaagatc tgctccaagc 3060

ctcggccgaa gatgatgtca aagcgataaa agacctcctc gaccagacta acaacctctt 3120

gcataagctg aagatttttc acatcagcca atccgaggat aaagctaaca tacttgacaa 3180

ggacgagcat ttctacttgg tcttcgagga gtgctacttt gagctggcta atatagtccc 3240

tttgtacaat aaaatacgga actatattac ccaaaaaccg tattctgatg aaaaattcaa 3300

gctcaacttc gaaaactcca cgctggccaa cggatgggat aagaacaaag aaccggacaa 3360

tacggccata cttttcataa aggacgacaa gtattatctc ggcgtcatga acaagaagaa 3420

caataaaatt ttcgacgaca aggcgatcaa agaaaataag ggcgaaggct ataagaagat 3480

cgtgtacaag ctgcttccgg gcgcaaacaa gatgctcccg aaagtatttt tcagcgccaa 3540

gtcgattaaa ttttacaacc ccagcgagga tattcttcgt atccgcaacc atagtactca 3600

cactaagaac ggttcgcctc agaaggggta cgagaaattt gaattcaaca ttgaggactg 3660

tcgcaagttc attgattttt acaagcagtc gatatcgaaa catccggagt ggaaggactt 3720

tggcttccgg ttcagtgata cccaaaggta caactcgatt gatgagtttt acagggaggt 3780

cgagaatcag ggatataagc tcacgttcga aaacatctcg gagtcataca tcgattcagt 3840

ggtcaaccaa gggaaacttt atttgtttca aatttacaac aaagattttt cagcgtactc 3900

gaagggccgc ccaaaccttc atactctgta ttggaaagcg ctcttcgacg agaggaatct 3960

ccaagacgtc gtgtataagc tgaacggaga agctgaactc ttttatcgca agcaatccat 4020

ccctaagaag ataacccacc cagcaaagga ggccatagcc aataagaata aggacaaccc 4080

gaaaaaggag agcgtgtttg aatacgacct cattaaggac aaacggttca ccgaggataa 4140

gttttttttt cactgcccta ttaccattaa cttcaaaagc tccggcgcga acaaatttaa 4200

cgatgaaatt aacctcctgc ttaaagagaa ggccaatgat gtgcacattt tgtctattga 4260

tagaggggaa aggcatcttg catactatac actcgtggac ggtaaaggga atataattaa 4320

gcaggatacc tttaacatca tagggaacga ccgtatgaag actaactatc acgataagct 4380

ggcagctatt gagaaggacc gcgattcggc acgtaaggac tggaagaaga ttaacaacat 4440

caaagagatg aaagaaggct atctctctca ggtcgtacac gagattgcca agctcgtgat 4500

agagtacaac gctattgtgg ttttcgaaga tttgaacttt ggctttaaaa ggggcaggtt 4560

caaagtggaa aagcaggtgt atcaaaagct ggagaaaatg ttgattgaga agctcaatta 4620

tctggtgttc aaagacaatg agtttgacaa gacgggaggt gtgctccggg cataccagct 4680

cactgcaccc ttcgagactt tcaaaaaaat ggggaagcag acaggaatta tttattacgt 4740

ccctgctggc ttcacaagca agatctgccc cgtaacggga ttcgtcaacc agctgtaccc 4800

aaagtatgaa tctgtctcca aaagtcagga attcttctcg aagttcgaca agatatgcta 4860

taatctcgat aagggctact ttgagttctc tttcgattac aagaactttg gggataaagc 4920

cgcgaagggt aaatggacta tagcgtcctt tgggtcgcgc ttgataaatt tccggaatag 4980

cgataaaaat cacaactggg acacccgcga agtctatcct acaaaagaac ttgaaaaact 5040

ccttaaggat tacagcatcg aatacgggca cggcgagtgt atcaaagcgg cgatctgtgg 5100

cgagtccgat aaaaagttct ttgcgaagct cacatcagtc ctgaatacga tcttgcagat 5160

gcggaactct aaaactggga cagagctcga ttacctgatc agtcctgtgg ccgacgttaa 5220

cgggaacttc tttgactcgc ggcaggcgcc aaagaacatg ccacaggatg cggatgccaa 5280

cggggcctat catattggcc tcaagggtct catgctcctg ggaagaatta agaataatca 5340

agaaggcaaa aaactcaatc tcgtaattaa gaatgaggaa tacttcgagt tcgttcagaa 5400

ccgtaacaat aagcgtccgg cagcgaccaa gaaggccgga caggctaaaa agaagaaggg 5460

aagttaccct tatgacgttc ctgattacgc gtatccttat gacgtcccgg actatgctta 5520

cccttacgat gttccagatt acgcctaacc cgggaattcg gtacgctgaa atcaccagtc 5580

tctctctaca aatctatctc tctctatttt ctccataaat aatgtgtgag tagtttcccg 5640

ataagggaaa ttagggttct tatagggttt cgctcatgtg ttgagcatat aagaaaccct 5700

tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca 5760

aaatccagta ctaaaatcca gatctcctaa agtccctata gatctttgtc gtgaatataa 5820

accagacacg agacgactaa acctggagcc cagacgccgt tcgaagctag aagtaccgct 5880

taggcaggag gccgttaggg aaaagatgct aaggcagggt tggttacgtt gactcccccg 5940

taggtttggt ttaaatatga tgaagtggac ggaaggaagg aggaagacaa ggaaggataa 6000

ggttgcaggc cctgtgcaag gtaagaagat ggaaatttga tagaggtacg ctactatact 6060

tatactatac gctaagggaa tgcttgtatt tataccctat accccctaat aaccccttat 6120

caatttaaga aataatccgc ataagccccc gcttaaaaat tggtatcaga gccatgaata 6180

ggtctatgac caaaactcaa gaggataaaa cctcaccaaa atacgaaaga gttcttaact 6240

ctaaagataa aagatctttc aagatcaaaa ctagagtcga cctgcaggca tgcaagcttg 6300

gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 6360

cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 6420

cgcccttccc aacagttgcg cagcctgaat ggcgaatgct agagcagctt gagcttggat 6480

cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt ttgacaggat atattggcgg 6540

gtaaacctaa gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag 6600

gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa 6660

gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc 6720

cgtcttctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc 6780

ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact 6840

agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc 6900

gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct 6960

gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca 7020

ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg 7080

cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc 7140

tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga 7200

ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg 7260

ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg 7320

cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg 7380

ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag 7440

tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg 7500

acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga 7560

cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg 7620

gtacgtgttc gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg 7680

tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg 7740

ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc 7800

gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct 7860

gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc 7920

ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc 7980

gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg 8040

attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc 8100

caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg 8160

cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc 8220

attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc 8280

acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag 8340

tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt 8400

gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa 8460

tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa 8520

gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca 8580

cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga 8640

tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc 8700

taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc 8760

ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga 8820

acgggcggtt ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga 8880

acccccaagc ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg 8940

cgcggcgctg ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca 9000

acgcatcgag gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg 9060

caaagaatcc cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg 9120

cgacgagcaa ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg 9180

cagcatcatg gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt 9240

gatccgctac gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc 9300

cagtgtgtgg gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa 9360

ccgataccgg gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga 9420

cgtactcaag ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac 9480

ctgcattcgg ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg 9540

ccgcctggtg acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag 9600

cgaaaccggg cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat 9660

cacagaaggc aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc 9720

cggcatcggc cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag 9780

atggttgttc aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg 9840

tttcaccgtg cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga 9900

ggcggggcag gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc 9960

atccgccggt tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa 10020

aggtcgaaaa ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat 10080

tgggaaccgg aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat 10140

gtaagtgact gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact 10200

tattaaaact cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga 10260

agagctgcaa aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg 10320

tcggcctatc gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc 10380

agggcgcgga caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct 10440

gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 10500

tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 10560

gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata 10620

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 10680

aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct 10740

cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 10800

ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 10860

ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 10920

cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 10980

actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 11040

cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 11100

tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 11160

gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 11220

caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 11280

agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 11340

tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 11400

tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 11460

gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 11520

gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta 11580

ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc 11640

cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg 11700

gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa 11760

gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa 11820

gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt 11880

aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa 11940

gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc 12000

gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg 12060

ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct 12120

ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca 12180

tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt 12240

catttttaaa tataggtttt cattttctcc caccagctta tataccttag caggagacat 12300

tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat 12360

tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa 12420

gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa 12480

taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg 12540

gagccgattt tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa 12600

catgctaccc tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc 12660

cgaatagcat cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc 12720

cgtcccggac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg 12780

ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac 12840

aacttaataa cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg 12900

ggatctggat tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt 12960

attttacaaa tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc 13020

ctataggaac cctaattccc ttatctggga actactcaca cattattatg gagaaactcg 13080

agcttgtcga tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg 13140

ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc 13200

gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg 13260

tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag 13320

agttggtcaa gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc 13380

ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag 13440

aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg 13500

accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg 13560

aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg 13620

acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca 13680

atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg 13740

ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc 13800

ggttgtagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca 13860

cggcgggaga tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga 13920

atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg 13980

gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga 14040

gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga 14100

aacttctcga cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga 14160

tctgcgaaag ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct 14220

ctccaaatga aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt 14280

gcgtcatccc ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt 14340

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 14400

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 14460

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 14520

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 14580

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat 14640

caatccactt gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg 14700

ggtgggggtc catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct 14760

ttatcgcaat gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag 14820

tgacagatag ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa 14880

gtctcaatag ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga 14940

gtgtcgtgct ccaccatgtt ggcaagctgc tctagccaat acgcaaaccg cctctccccg 15000

cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 15060

gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 15120

ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 15180

acagctatga ccatgattac g 15201

<210> 17

<211> 692

<212> DNA

<213> Artificial sequence (crRNA)

<400> 17

gatccaagct tcgttgaaca acggaaactc gacttgcctt ccgcacaata catcatttct 60

tcttagcttt ttttcttctt cttcgttcat acagtttttt tttgtttatc agcttacatt 120

ttcttgaacc gtagctttcg ttttcttctt tttaactttc cattcggagt ttttgtatct 180

tgtttcatag tttgtcccag gattagaatg attaggcatc gaaccttcaa gaatttgatt 240

gaataaaaca tcttcattct taagatatga agataatctt caaaaggccc ctgggaatct 300

gaaagaagag aagcaggccc atttatatgg gaaagaacaa tagtatttct tatataggcc 360

catttaagtt gaaaacaatc ttcaaaagtc ccacatcgct tagataagaa aacgaagctg 420

agtttatata cagctagagt cgaagtagtg attgtaattt ctactaagtg tagatctatg 480

ccagctcaag ttcaatgcat aatttctact aagtgtagat gaggcagtga atcaatgggc 540

catttaattt ctactaagtg tagatgttct tctccgcgtc cagctaaact aatttctact 600

aagtgtagat cagtcgataa acccgccaaa cctgtaattt ctactaagtg tagatcacct 660

aaaacatctc ttgcttcaat ttttttggta cc 692

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence (primer set F)

<400> 18

aactgggtat gcgaagactt cc 22

<210> 19

<211> 26

<212> DNA

<213> Artificial sequence (primer set R)

<400> 19

aagcaagcac aagagtgtgt gtcaac 26

Claims (3)

1. An application of a CRISPR/Cas12a gene editing system in 84K poplar gene editing comprises the following steps:

   step (1): selection of 84K poplar phytoene dehydrogenase gene 8 target site

   Extracting wild poplar '84K' gene DNA according to a CTAB method, and cloning phytoene dehydrogenase gene 8 from the extracted poplar '84K' gene DNA;

   amplifying PagPDS by using a specific primer F and a specific primer R; the PCR products were electrophoresed on a 1% agarose gel; DNA was recovered using the TIANgel Midi purification kit and cloned into the pMD18-T Simple vector;

   step (2): construction of vectors

   Designing 5 gRNAs, namely gRNA1, gRNA2, gRNA3, gRNA4 and gRNA5 according to an NGG design principle, connecting the 5 gRNAs and tRNA-crRNA in series, wherein each unit comprises a mature direct repeat and a guide sequence of 24bp, and a plurality of crRNA cassettes driven by an Arabidopsis RNA polymerase III promoter AtU6-26, a 2x35S promoter in a pCambia1300 binary vector drives AsCas12a, lbCas12a and FnCas12a, editing is carried out on the AsCas12a, the LbCas12a or the FnCas12a, and the constructed vectors are named as PagPDS-AsCas12a, pagPDS-LbCas12a and PagPDS-FnCas12a respectively;

   and (3): genetic transformation of poplar

   The expression vector is transferred into agrobacterium GV3101 by an electric shock method, and the vector is transferred into poplar by agrobacterium mediation, and the transformation steps are as follows: the 84K tissue culture seedling of the hybrid poplar clone used for genetic transformation is cultured under the conditions that the culture temperature is 23-25 ℃, the illumination is 16/8h and the illumination intensity is 50 mu Mm-2 s-1;

   infecting an 84K leaf disc by agrobacterium containing a carrier when OD600= 0.6-0.8, co-culturing the infected leaf disc on an adventitious bud induction culture medium for 3 days under a dark condition at the temperature of 22 +/-2 ℃, transferring the co-cultured leaf disc to SIM containing 3mg/L hygromycin B and 200mg/L Timentin, inducing and screening resistant adventitious buds under the conditions of the culture temperature of 23-25 ℃, the illumination of 16/8h and the illumination intensity of 50 mu Mm-2s-1, transferring the resistant adventitious buds to a rooting culture medium containing 3mg/L hygromycin B and 200mg/LTimentin after about 30 days of induction culture until adventitious roots are induced, extracting rooted leaf DNA, and carrying out PCR verification;

   and (4): obtaining transgenic plants

   30 transgenic plants with positive resistance screening are respectively obtained by a PagPDS-AsCas12a, a PagPDS-FnCas12a and a PagPDS-LbCas12a through an agrobacterium transformation leaf disc method, and biallelic homozygous mutant and heterozygous mutant of PDS show albino phenotype;

   and (5): directional editing condition analysis of CRISPR system on poplar genes

   Extracting gene DNA from stable transgenic plants and wild-type plants by using a CTAB method, using the gene DNA as a template, carrying out PCR amplification on an endogenous PagPDS fragment through a primer pair sequence F and a primer pair sequence R, covering a region containing five target sites of a Cas12a system by using primers, separating, recovering and cloning a PCR product into a pMD18-T Simple vector on 1% agarose gel, identifying mutation by carrying out Sanger sequencing on a single clone, and comparing all sequence results with a reference sequence of PagPDS by carrying out comparison in SnapGene;

   and (6): heat treatment of callus cultured after infection with Agrobacterium

   Firstly, culturing a transformation material in the dark for 2 days, cleaning agrobacterium, transferring the material to an induction callus culture medium, inducing callus in the dark at room temperature for 4 weeks, then carrying out heat treatment on the callus at 28 ℃, culturing at 28 ℃ for 30h, transferring to 22 ℃ for culturing for 42h, repeating for 4 times, standing at room temperature for normal culture, transferring the material to an induction budding culture medium for induction budding, and recording the morphology and the molecular phenotype.
2. 2. The use of the CRISPR/Cas12a gene editing system according to claim 1 in 84K poplar gene editing, characterized in that: the adventitious bud induction culture medium in the step (3) is as follows: SIM, murashige-Skoog minimal medium supplemented with 0.5mg/l 6-benzylaminopurine and 0.05mg/l naphthalene acetic acid.
3. 3. The use of the CRISPR/Cas12a gene editing system according to claim 2 in 84K poplar gene editing, characterized in that: the rooting culture medium in the step (3) is as follows: RIM,1/2MS minimal medium supplemented with 0.05mg/L IBA and 0.02mg/L NAA.

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