CN110747186B - CRISPR/Cas9 systems and methods for efficient generation of mutants not carrying a transgenic element in plants - Google Patents

Abstract

The present invention relates to a construct for CRISPR/Cas9 gene editing in plants to efficiently produce gene-edited mutant plants that do not carry a transgenic element. Also relates to systems and methods for CRISPR/Cas9 gene editing of plants using the constructs.

Description

CRISPR/Cas9 systems and methods for efficient generation of mutants not carrying a transgenic element in plants

Technical Field

The invention belongs to the field of plant gene editing. In particular, the present invention relates to a self-cleaving peptide-based construct for CRISPR/Cas9 gene editing in plants, and systems and methods for using the construct to efficiently obtain mutants that are edited and do not carry a transgenic element. The invention further provides a construct for CRISPR/Cas9 multiplex gene editing in plants, and a system and method for efficiently obtaining a multiplex-edited mutant without carrying a transgenic element using the construct.

Background

A CRISPR/CRISPR-associated (Cas) genome editing system is an accurate gene editing system and is widely used for researching functional genomes of animals, plants and the like in recent years. The conventional CRISPR/Cas9 system contains two components, a single guide RNA (sgRNA) for recognizing and matching the target DNA and a Cas9 endonuclease for cleaving the target DNA.

After gene editing of a plant using CRISPR/Cas9 technology, the CRISPR/Cas9 expression element remaining in the plant genome would allow the edited plant to fall into the category of transgenic plants. In practice it is often desirable that the edited organism does not eventually carry any foreign elements for gene editing, in other words it is desirable to obtain a non-transgenic organism. In agriculture and commerce, non-transgenic plants are favored, are easier to examine and approve, can be widely applied and realize higher value. However, unlike animals, it is more difficult to obtain plants that do not contain a CRISPR/Cas9 expression element. In animals, the rapid and efficient acquisition of multiple site-edited non-transgenic animals can be achieved by injecting multiple sgrnas and transcripts of Cas9 into fertilized eggs simultaneously. Editing in plants is typically performed on plant tissues such as callus or germ cells, for example by introducing the CRISPR/Cas9 expression element into the plant by means of agrobacterium infection. Therefore, when a plant which is subjected to gene editing and does not contain a CRISPR/Cas9 expression element is obtained, after the target gene is edited by the CRISPR/Cas9, the CRISPR/Cas9 element is separated by utilizing the Mendel's separation law and the genotype identification is adopted, so that the method is time-consuming and labor-consuming.

In addition to the desire to obtain non-transgenic plants, there is also a need in the art to remove the CRISPR/Cas9 element in edited plants. If the CRISPR/Cas9 element is not removed, the CRISPR/Cas9 element is left in the plant body, so that on one hand, the source of the mutation is difficult to distinguish, and the stable heritable mutation is difficult to obtain; on the other hand, increases the risk of off-target. In particular, in case Cas9 and sgRNA are still present in mutant plants generated by the CRISPR/Cas9 system, it will be difficult to distinguish whether a certain mutation is inherited from the previous generation or newly generated in the current generation, which is clearly important since the newly generated mutation in the current generation may be an epigenetic somatic mutation. In addition, editing elements used in CRISPR systems, including Cas9 and sgrnas, are present in a constitutive expression manner, and if present in the T2 generation or even in subsequent generations, off-target mutations may be generated by reediting an already mutated target sequence or editing other similar sequences of the genome via sgRNA mismatches. In addition, if the CRISPR/Cas9 element remains in the plant, complementation verification of gene function cannot be performed, because the newly transferred gene sequence is edited by the CRISPR/Cas9 element. Meanwhile, the plant containing the CRISPR/Cas9 element remains as a generally regarded transgenic plant and is difficult to be approved for commercial growth applications.

Therefore, developing a method for editing a plant specific gene that can simply screen a transgene element not carrying CRISPR/Cas9 is important for plant functional gene research and agricultural production.

Recently, a mCherry fluorescence based Cas-free 9 system was reported, wherein mCherry fluorescence is specifically expressed in seeds of Arabidopsis under the control of the At2S3 promoter, whereby seeds of T2 generation that do not contain the Cas9 transgene can be selected by visual inspection of the presence of fluorescence (Gao et al, Plant Physiology 171, 1794-. This system facilitates the selection of Cas-free 9 plants. However, in this system, Cas9 and mCherry are still present in two separate expression cassettes, driven by different promoters, and thus the emitted fluorescence intensity is not correlated with the expression level of Cas9, and cannot be used to predict the efficiency of gene editing.

Platt et al (Platt et al, Cell 159,440-455,2014) in its article on the mouse model proposed a Cas9-P2A-EGFP construct for mice that visualizes cells expressing Cas9 by enhancing green fluorescent protein and that exploits the self-cleaving properties of the 2A peptide, but this study does not involve any application in plants.

Petersen et al (Petersen et al, BMC Biotechnol 19,36,2019) used an Agrobacterium-mediated delivery system to deliver viral replicons to express GFP-labeled rRNA/Cas9, in combination with fluorescence-activated cell sorting (FACS) to increase editing efficiency. The use of Cas9-2A-GFP expression cassettes driven by the CaMV35S promoter in tobacco is mentioned, but it is not clear what source of 2A peptide is used. Since this article focuses on the combination of GFP and FACS sorting, it is limited to transformation and gene editing of partial tissues of plants, and does not discuss editing efficiency and removal of editing elements at the level of whole plant plants, nor does it study the effect of editing on T2 generation plants.

Therefore, there is still a need in the art for a universal CRISPR/Cas9 system suitable for plant gene editing to efficiently and easily generate and screen mutant plants that are efficiently edited but do not carry transgenic elements.

Disclosure of Invention

To solve the above problems, the inventors of the present invention developed a self-cleaving peptide-based construct for plant gene editing, specifically comprising a Cas9 expression cassette of the structure "Cas 9-P2A-reporter", wherein P2A represents the porcine teschovirus 2A peptide. The inventor finds that when the P2A peptide is coupled with Cas9 protein and reporter molecule GFP and used for plant gene editing, the self-cleavage efficiency is as high as 95%, the self-cleavage efficiency is obviously superior to that of 2A peptide from other sources, and the coupled reporter molecule is ensured not to influence the cleavage activity of the Cas9 protein in the gene editing; meanwhile, as the Cas9 protein and the reporter molecule are in the same expression cassette and are transcribed together under the regulation and control of the same promoter, the Cas9 protein and the reporter molecule are always expressed in equal stoichiometric amount, so that the fluorescence intensity of the GFP of the reporter molecule can indicate the protein level of Cas9, and the protein level of Cas9 and the gene editing efficiency present a certain positive correlation, so that the gene editing efficiency in the first-generation plant can be simply judged through the reporter molecule; second generation plants can also be screened for those not carrying the transgenic element by the absence or presence of the reporter molecule in order to obtain mutants not carrying the transgenic element. The inventors experimentally verified the efficacy of the editing system of the invention in acting on different target genes, wherein the construct comprising the Cas9 expression cassette of the invention, when used for CRISPR/Cas9 gene editing in plants, is capable of efficiently producing and selecting gene-edited T1 generation plants and also facilitates the selection of gene-edited T2 generation plants that do not carry a transgenic element. On the basis, the inventor also combines the construct with a strategy for constructing multiple sgrnas based on isocaudarner, so that efficient multiple gene editing is realized, and the construct is proved to be also suitable for the application of editing multiple genes simultaneously. The present invention has been completed based on the above findings.

Thus, in a first aspect, the present invention relates to a construct of a CRISPR/Cas9 gene editing system for plants comprising a Cas9 expression cassette, said Cas9 expression cassette comprising, from 5 'to 3': (a) a promoter for a Cas9 expression cassette, (b) a nuclease coding sequence, (c) a self-cleaving peptide coding sequence, and (d) a reporter gene sequence, wherein (a) and (b) - (d) are operably linked such that the nuclease coding sequence and reporter gene sequence are under the control of the promoter, the nuclease coding sequence encodes Cas9 or a fragment or variant thereof having DNA cleaving activity, and the self-cleaving peptide coding sequence encodes a 2A peptide derived from porcine scheimpfera virus.

In one embodiment, the construct further comprises a sgRNA module comprising one or more sgRNA expression cassettes, wherein each sgRNA expression cassette comprises, from 5 'to 3': (e) a promoter for the sgRNA expression cassette; and (f) sgrnas for target DNA sequences to be edited. When the sgRNA module comprises multiple sgRNA expression cassettes, it is used for multiplex gene editing.

In a second aspect, the present invention relates to a method of making a construct for CRISPR/Cas9 gene editing in a plant comprising introducing into a vector a Cas9 expression cassette, said Cas9 expression cassette comprising, from 5 'to 3': (a) a promoter for a Cas9 expression cassette, (b) a nuclease coding sequence, (c) a self-cleaving peptide coding sequence, and (d) a reporter gene sequence, wherein (a) and (b) - (d) are operably linked such that the nuclease coding sequence and reporter gene sequence are under the control of the promoter, the nuclease coding sequence encodes Cas9 or a fragment or variant thereof having DNA cleaving activity, and the self-cleaving peptide coding sequence encodes a 2A peptide derived from porcine scheimpfera virus.

In a preferred embodiment, the construct preparation method of the invention further comprises introducing into the vector a sgRNA module comprising one or more sgRNA expression cassettes, wherein each sgRNA expression cassette comprises from 5 'to 3': (e) a promoter for the sgRNA expression cassette; and (f) sgrnas against the labeled target DNA sequences.

In a preferred embodiment, the sgRNA module comprising a plurality of sgRNA expression cassettes used in the construct preparation method of the present invention is obtained by assembling together a plurality of sgRNA expression cassettes, said assembling comprising adding upstream and downstream restriction enzymes, respectively, upstream and downstream of each sgRNA expression cassette, said upstream and downstream restriction enzymes being different restriction enzymes and being mutually identical enzymes, wherein the upstream restriction enzyme added for all sgRNA expression cassettes is identical and the downstream restriction enzyme added for all sgRNA expression cassettes is identical.

In specific embodiments, the isocaudarner is Spe I and Nhe I.

In a third aspect, the present invention relates to a construct obtained by the method of the second aspect.

In a fourth aspect, the present invention relates to a method of gene editing in a plant comprising the use of a construct according to the first or third aspect of the invention.

In a fifth aspect, the present invention relates to a method for obtaining a mutant not carrying a transgenic element in a plant, comprising: (i) transforming a plant with the construct of claim 6 or 10; (ii) selecting a first generation plant with positive reporter gene expression as a transformant; (iii) (iii) passaging the first generation plants in (ii) to obtain second generation plants; and (iv) selecting second generation plants that are negative for reporter gene expression as mutants that have undergone gene editing and do not carry a transgenic element.

In a preferred embodiment of the fifth aspect, after selecting a plant by reporter gene expression in steps (ii) and (iv), the target gene sequence of the selected plant is identified to confirm that it is edited. In particular embodiments, the identification is by sequencing, for example by Sanger sequencing.

In a preferred embodiment of each aspect of the invention, the self-cleaving peptide encoding sequence is the nucleotide sequence set forth in SEQ ID NO. 5 or a mutant thereof encoding the same polypeptide. In alternative embodiments, the self-cleaving pseudosequence is a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homology to the nucleotide sequence of SEQ ID No. 5 and encodes an amino acid sequence identical to the amino acid sequence encoded by SEQ ID No. 5. In another embodiment, the self-cleaving peptide coding sequence is a nucleotide sequence that hybridizes under stringent conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, with SEQ ID NO. 5.

In a preferred embodiment of the various aspects of the invention, the Cas9 of the invention is Cas9(SpCas9) derived from streptococcus pyogenes.

In a preferred embodiment of the various aspects of the invention, the reporter gene comprised in the Cas9 expression cassette of the invention encodes a protein selected from the group consisting of: luciferase, green fluorescent protein, red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein.

In a preferred embodiment of the various aspects of the invention, the promoter comprised in the Cas9 expression cassette of the invention is a promoter that is constitutively expressed in plants. Preferably, the promoter is an arabidopsis ubiquitin 10 gene (AtUBQ10) promoter or a cauliflower mosaic virus (CaMV)35S promoter. Most preferably, the promoter is the AtUBQ10 promoter.

In preferred embodiments of the various aspects of the invention, the Cas9 expression cassette of the invention comprises a Nuclear Localization Signal (NLS) coding sequence flanking both nuclease coding sequences.

In a preferred embodiment of the various aspects of the invention, the construct is constructed based on vectors commonly used in plants. Preferably, the vector is a binary vector.

The editing system has the advantages that the mutant plants which are edited but do not carry the transgenic elements can be simply, conveniently and efficiently generated and screened; editing of multiple genes can be achieved by combining the expression cassettes of the invention with a coterminal enzyme and obtaining multiple mutant plants that do not carry transgenic elements. On the other hand, the plants generated by the present invention, which do not carry the CRISPR/Cas9 transgenic element, make it possible to develop complementation experiments (complementation experiments), because if the CRISPR/Cas9 transgenic element in the edited plant is not removed, the wild-type gene used for complementation experiments may also be edited, so that the gene-phenotype association cannot be established by complementation experiments.

Drawings

FIGS. 1A-E relate to the process of efficiently generating transgenic element-free and multiple gene-edited mutants in Arabidopsis. FIGS. 1A-C present the experimental results obtained in example 1 using 5 2A peptides. Figure 1A is a photograph of protoplasts showing the distribution of fluorescence generated when the construct "Cas 9-linker-GFP" is expressed in arabidopsis mesophyll protoplasts ("linker" is a control peptide or a different 2A peptide). Three columns from left to right are: GFP channel, bright field and merged images. From top to bottom: empty plasmid (Empty), GFP control (GFP), Control Peptide (CP), and 5 different sources of 2A peptide, i.e., F2A-1, E2A, F2A-2, P2A, and T2A. FIG. 1B is the result of immunoblot analysis of total protein extracted from Arabidopsis mesophyll protoplasts as shown in FIG. 1A and Western blot analysis using GFP antibody, which demonstrates the cleavage efficiency of different 2A peptides. Figure 1C is the results of quantifying the lysis efficiency shown in figure 1B using Image J mapping software, the results are from five independent experiments, showing the data as mean ± SEM. Fig. 1D is a schematic diagram of construction of sgRNA modules comprising multiple sgRNA expression cassettes using a isocaudarner. Fig. 1E demonstrates an example of a vector in the CRISPR/Cas9 system of the invention. Wherein the Cas9-P2A-GFP expression cassette is driven by the AtUBQ10 promoter or the dual CaMV35S promoter, and rbcS-E9 is used as a terminator. The assembled multiple sgRNA modules comprising multiple sgRNA expression cassettes are inserted into a binary vector by means of the restriction enzymes Kpn I and Sal I. HygR, hygromycin resistance gene; GmR, gentamicin sulfate resistance gene; KanR, kanamycin resistance gene; SmR, spectinomycin resistance gene. HygR and KanR belong to the pCambia1300-Cas9-P2A-GFP binary vector construct, while GmR and SmR belong to the pJim19-Cas9-P2A-GFP binary vector construct.

FIGS. 2A-D illustrate the procedure for selecting plants that do not carry a transgenic element, as shown in example 3. FIG. 2A is an exemplary workflow diagram of the editing system of the present invention. Wherein a construct with the target sgRNA expression cassette and Cas9-P2A-GFP is delivered into arabidopsis thaliana by agrobacterium-mediated transformation, seeds from transformed plants are sown on MS plates with antibiotics (hygromycin or gentamicin sulfate in the figure, it will be understood by those skilled in the art that the corresponding antibiotics should be used in the selection medium based on the vector chosen). T1 transgenic plants with high GFP expression were picked, whereas T2 transgenic plants without GFP expression (plants not carrying the transgenic elements) were selected. Genotyping was performed to determine the presence of the target gene in T1 plants and those carrying no transgenic elements in T2. FIG. 2B shows the isolation of T2 transgenic plants with or without a GFP signal. Four sgrnas were assembled, two targeting BZR1 and two targeting BES1, using the method described in fig. 1D, and cloned into the binary vector P35S: Cas9-P2A-gfp (gent). The resulting constructs were transferred into wild type to edit BZR1 and BES 1. 3 days of light growth seedlings were photographed using a confocal microscope. The number in the lower right hand corner of each panel is the number of plants observed. Figure 2C is an agarose gel analysis for Cas9-P2A-GFP expression cassette. Three T2 plants without GFP signal and nine T2 plants with GFP signal were randomly selected from the plants of fig. 2B, PCR products were amplified using primers for Cas9, and analyzed by agarose gel to confirm the accuracy of fluorescence-based selection. FIG. 2D is the DNA sequence of the target site of BZR1 and BES1 in representative T2 transgenic plants without GFP expression (accession numbers 10-19). BZR1 locus 1 and BZR1 locus 2 are two genomic sites comprising BZR1sgRNA, while BES1 locus 1 and BES1 locus 2 are two genomic sites comprising BES1 sgRNA. The sgRNA sequence is underlined. Plus signs indicate insertions and minus signs indicate deletions.

Figures 3A-D relate to the process of editing a genome using Cas9-P2A-GFP, as shown in example 2. FIGS. 3A and 3C are photographs of plants showing that the fluorescence intensity of transgenic plants correlates with the phenotype resulting from editing BRI1, with the leftmost column being a photograph of a 4-day-old T1 young plant, the second column being a photograph of a 4-day-old T1 young plant (taken using a Leica fluoroscope), and the remaining right part being a photograph of the plant after several weeks of growth in soil. Cas9-P2A-GFP is driven by the dual CaMV35S promoter (A) or the AtUBQ10 promoter (C). Fig. 3B and 3D are Sanger sequencing results of the representative T1 transgenic line in fig. 3A and 3C. The BRI1sgRNA sequence is underlined, with the three nucleotides AGG in the box after the underlining being the original spacer adjacent motif (PAM) sequence.

FIG. 4 illustrates the construction flow of plasmids for genome multiplex editing and selection of multiplex edited plants not carrying a transgenic element. Primers for sgrnas were annealed and inserted between AtU6-26 and the scaffold using Bbs I. Multiple sgRNA expression cassettes were assembled using Spe I/Sal I and Nhe I/Sal I. The tandem sgRNA expression cassette was digested with Kpn I and Sal I and then cloned into a binary vector with Cas9-P2A-GFP expression cassette. Cas9-P2A-GFP is driven by the dual 35S promoter or the AtUBQ10 promoter and is terminated by the rbcS-E9 terminator.

Fig. 5 shows the sequence of vector pAtU6-26-M for sgRNA. The first italic bold sequence is the arabidopsis AtU6-26 promoter, the second italic bold sequence is the sgRNA scaffold. Each restriction enzyme site is underlined and the corresponding restriction enzyme is labeled. The corresponding sgRNA primer sequences are also shown.

FIGS. 6A-D show that multiple target sites were efficiently edited in T1 transgenic plants with high GFP expression. FIG. 6A shows GFP signals in T1 transgenic plants generated after multiple genome editing by the system of the present invention. The multiplex editing uses four sgRNA expression modules, two for targeting BZR1 and two for targeting BES1, assembled together by a co-tailase and inserted into P35S: Cas9-P2A-gfp (hyg). The final binary vector was transferred to Agrobacterium and delivered to Col-0 plants by floral dip. Fluorescence signals were collected from 5 day old light-grown seedlings by LSM 710 confocal microscopy. Two representative seedlings (#1 and #10) with strong GFP fluorescence are shown. FIGS. 6B-D are DNA sequencing diagrams of the T1 transgenic line in FIG. 6A. Locus 1 and locus 2 are the genomic loci to which BZR1 sgrnas are directed, while locus 3 and locus 4 are the genomic loci to which BES1sgRNA is directed. sgRNA is a 20bp sequence with a start site G, shown underlined.

FIGS. 7A-B show detection of Cas9-P2A-GFP in T2 plants. FIG. 7A shows the self-cleavage efficiency of the P2A peptide when used to edit the Arabidopsis BZR1 and BES1 genes. T2 transgenic plants with GFP fluorescence were from T1 lines (#1 and #10) described in fig. 2B and 2C. Total protein was extracted from 5 day old light grown seedlings and analyzed by immunoblotting using GFP antibody. Asterisks indicate a nonspecific band; RPN6 was a loading control. Figure 7B is a PCR and agarose gel analysis of Cas9-P2A-GFP DNA expression cassettes in plants without GFP fluorescence. DNA from T2 plants without GFP fluorescence was extracted and amplified using specific primers for Cas 9. "C" indicates a positive control.

Detailed Description

The practice of some of the methods disclosed herein, unless otherwise indicated, employs conventional techniques of botany, biochemistry, chemistry, molecular biology, cell biology, genetics and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: a Laboratory Manual, 4th Edition (2012), and the like.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or a standard deviation of greater than 1, according to practice in the art. Alternatively, "about" may represent a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may denote an order of magnitude, i.e. 10 times, preferably within 5 times, more preferably within 2 times the value. Where particular values are described in the application and claims, the term "about" should be understood to be within an acceptable error range in this context unless otherwise indicated.

The term "gene" as used herein refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence encoding an RNA transcript. As used herein, the term with respect to genomic DNA includes intervening non-coding regions as well as regulatory regions, and may include 5 'and 3' ends. In some uses, the term includes transcribed sequences, including 5 'and 3' untranslated regions (5'-UTR and 3' -UTR), exons and introns. In some genes, the transcribed region will comprise an "open reading frame" encoding the polypeptide. In some uses of this term, a "gene" comprises only coding sequences (e.g., "open reading frames" or "coding regions") necessary to encode a polypeptide. In some cases, the gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (trna) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in a native location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises a mutation, insertion, and/or deletion (e.g., a non-native sequence).

The term "nucleotide" as used herein generally refers to an alkali-sugar-phosphate combination. The nucleotides may comprise synthetic nucleotides. The nucleotides may comprise synthetic nucleotide analogs. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside Adenosine Triphosphate (ATP), Uridine Triphosphate (UTP), Cytosine Triphosphate (CTP), Guanosine Triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. These derivatives may include, for example, [ α S ] dATP, 7-deaza-dGTP and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphate (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length, and can be in single-, double-or multi-stranded form. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar or nucleobases).

The term "homology" refers to the percentage of sequence similarity between two or more nucleotide sequences. Those skilled in the art know that homology between nucleotide sequences can be aligned and determined using a instrumental computer algorithm such as Blast with its default parameters.

The term "stringent conditions" herein refers to conditions under which hybridization experiments are performed, which may be low stringency conditions, medium stringency conditions or high stringency conditions, preferably high stringency conditions. For example, "low stringency conditions" can refer to conditions using 5x SSC, 5x Denhardt's solution, 0.5% SDS, and 52% formamide at 30 ℃; "moderately stringent conditions" may refer to conditions using 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, and 52% formamide at 40 ℃; "high stringency conditions" can refer to conditions using 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, and 52% formamide at 50 ℃. It will be appreciated by those skilled in the art that the homology between nucleotide sequences that are capable of hybridising to each other at higher temperatures is also higher.

The CRISP-Cas9 technology is a gene site-directed editing technology and is widely used for plant gene editing. The system mainly comprises a Cas9 protein for cutting DNA and a section of guide RNA. The guide RNA binds to Cas9 and recognizes and pairs with the DNA sequence of interest by approximately 20 nucleotides upstream thereof, bringing Cas9 to the region to be cleaved and cleaving double-stranded DNA. Cas9 protein can be derived from a variety of different species. Can be a Cas9 protein derived from a microorganism selected from the group consisting of: bifidobacterium species (Bifidobacterium spp.), curdlan species (Kandleria spp.), Lactobacillus species (Lactobacillus spp.), Leuconostoc spp., Oenococcus species (Leuconostoc spp.), Oenococcus species (Oenococcus spp.), Pediococcus species (Pediococcus spp.), Staphylococcus species (Staphylococcus spp.) such as Staphylococcus aureus (s. aureus), Streptococcus species (Streptococcus spp.) such as Streptococcus pyogenes (s. pyogenenes) and Weissella species (Weissella spp.). In preferred embodiments, the Cas9 protein is Cas9, i.e., spCas9, derived from streptococcus pyogenes or Cas9, i.e., saCas9, derived from staphylococcus aureus. Variants or fragments of Cas9 protein having DNA cleavage activity of Cas9 may also be used herein. The "coding sequence of Cas9 protein" of the present invention refers to a nucleotide sequence encoding a Cas9 protein or a variant or fragment thereof having DNA cleavage activity of Cas 9. In further embodiments, the coding sequence for Cas9 protein may be codon optimized for better expression in the plant to be edited. In particular embodiments, the Cas9 sequence is derived from a vector known in the art or commercially available, such as the hspscas 9 used in the literature of Feng et al (Feng et al, Cell Research 23,1229-1232, 2013).

"transgenic element" refers to a segment of foreign DNA of interest that causes a predictable, targeted genetic alteration in a recipient organism. The promoter of Cas9 protein, Cas9-P2A-GFP gene coding region, transcription termination element, promoter of sgRNA, transcription region and other related sequences derived from vector are mainly contained in the invention. In the context of the present invention, "transgenic element" is sometimes used interchangeably with "gene editing element".

"sgRNA" refers to a guide RNA for gene editing, including mature crRNA tracrRNA, which cleaves DNA double strand by complementary pairing of sgRNA and DNA of interest by bringing it together with Cas9 and transporting it to the DNA of interest. The principle and method of designing sgrnas from the target DNA to be edited are known to those skilled in the art. Examples of sgrnas are given herein for exemplary target genes, but this should not be construed as limiting the invention.

A "self-cleaving peptide", specifically a "2A peptide", is a peptide capable of self-cleavage. The 2A sequence typically encodes a short 18-22 amino acid peptide that mediates a translational "jump" from the glycyl-prolyl peptide bond near its 3' terminus (Donnelly et al, Journal of General Virology 82,1013-1025, 2001; de Felipe et al, Biotechnology Journal 5,213-223, 2010). 2A peptides are capable of efficient self-cleavage and their flanking proteins are capable of stoichiometric expression and have therefore been widely used in animal cells. However, the cleavage efficacy of the various 2A peptides in plant cells is not clear. The article by Kim et al (Kim et al, PLoS One 6, e18556,2011) mentions that although the 2A peptide can be used to construct polycistronic or bicistronic vectors and is smaller and more efficient to cleave than IRES (internal ribosome insertion site) commonly used for this purpose, it has not been widely used, with One impediment being that the cleavage efficiencies of the 2A peptides of various origins reported so far differ in different contexts. Kim et al compared the potency of four different sources of 2A peptide in human cell lines, zebrafish and mouse, but no relevant studies have been carried out in plants nor have been directed to the use of 2A peptide in combination with CRISPR-Cas9 system or fusion with Cas 9.

"reporter genes" refers to a class of genes that are expressed under specific conditions, thereby allowing them to produce traits that are easily detected and not otherwise produced by the test material. Reporter genes are generally useful for indicating the expression of other expression elements to which they are operably linked. In embodiments of the invention, since the reporter gene and the nuclease encoding gene, such as the Cas9 encoding gene, are under the control of the same promoter, the detectable signal generated by the reporter gene can be used to determine the expression of Cas 9. In some embodiments, the reporter gene encodes a reporter protein, such as an enzyme, including but not limited to galactosidase, luciferase, or alkaline phosphatase. In some embodiments, the reporter gene used to indicate nuclease expression encodes a fluorescent protein, such as Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Blue Fluorescent Protein (BFP), and Yellow Fluorescent Protein (YFP). Preferably, a reporter gene encoding a fluorescent protein, in particular a reporter gene encoding GFP, is used in the constructs of the invention.

An "expression cassette" in the context of the present invention refers to a piece of DNA, usually part of a certain vector or construct or to be introduced into a vector or construct, which usually comprises one or more coding sequences to be expressed and control sequences regulating the expression of said coding sequences, such as a promoter. One or more expression cassettes may be included in the construct, for example a construct of the invention may comprise one expression cassette for expressing Cas9, and one or more sgRNA expression cassettes. In the context of the present invention, a "Cas 9 expression cassette" generally refers to an expression cassette comprising a promoter and Cas9, while a "sgRNA expression cassette" generally refers to an expression cassette comprising a promoter and a sgRNA.

A "sgRNA module" refers in the context of the present invention to a sequence segment which may comprise one or more sgRNA expression cassettes.

"vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (the target gene) into a recipient cell. The three most commonly used vectors are bacterial plasmids, bacteriophages and animal and plant viruses. Binary vectors are preferably used in the present invention. Vectors that can be used in the present invention include, but are not limited to, pJim series vectors such as pJim19, etc., pCAMBIA series vectors such as pCAMBIA1301, etc.

"construct" or "expression construct" in the context of the present invention generally refers to a piece of DNA, usually circular or linear, that is vector-based and used for a particular function or purpose by the introduction of a fragment of foreign sequence.

A "promoter" is a DNA sequence recognized, bound and transcribed by RNA polymerase, which contains conserved sequences required for RNA polymerase specific binding and transcription initiation, most of which are located upstream of the transcription initiation point of a structural gene and are not generally transcribed per se. A variety of promoters suitable for use in plants are known, including constitutive promoters, inducible promoters, specific promoters such as tissue-specific or developmental stage-specific promoters. Multiple promoters will typically be included in the constructs for CRISPR/Cas9 of the invention, such as a promoter for driving the Cas9 expression cassette, and a promoter for driving the sgRNA expression cassette.

In embodiments of the invention, it is preferred to use a "constitutive promoter", i.e. a promoter capable of functioning in all tissues and promoting sustained expression. Constitutive promoters commonly used in plants include, for example, ubiquitin promoters such as maize ubiquitin promoter, Arabidopsis ubiquitin promoter, actin promoter, cauliflower mosaic virus promoter (CaMV 35S), and the like. However, it is also known to the skilled person that although a variety of commonly used constitutive promoters are known in the art, the performance of the promoters may vary for different plants, different tissues, different driven genes and different applications, and the applicability of the various promoters is often difficult to predict without extensive work. In a preferred embodiment of the invention, the promoter is the arabidopsis thaliana ubiquitin 10 gene promoter (AtUBQ10) or the cauliflower mosaic virus 35S promoter (CaMV 35S), more preferably the arabidopsis thaliana ubiquitin promoter. Accordingly, in the context of the present invention, "AtUBQ 10" or "UBQ 10" both refer to the arabidopsis thaliana ubiquitin 10 gene promoter, unless otherwise indicated.

"operably linked" refers to an arrangement of two or more elements that are functionally related such that each element functions normally and wherein at least one element (e.g., a transcriptional regulatory element, e.g., a promoter, a terminator, an enhancer) modulates the function of at least one of the remaining elements (e.g., a coding sequence, an open reading frame). For example, a promoter is operably linked to a coding sequence, indicating that the promoter is capable of regulating the level of transcription of the coding sequence. "operably linked" does not necessarily mean that two or more of the recited elements are directly adjacent.

The constructs, systems and methods of the invention are particularly useful for gene editing in plants. In the context of the present invention, "plant" includes monocotyledonous plants such as oil palm, sugarcane, banana, maize, wheat, rye, barley, oats, rice, millet or sorghum, and dicotyledonous plants such as Arabidopsis thaliana, soybean, potato, oilseed rape, peanut or sunflower. The plant of the invention can be a cash crop, a model plant, a flower and a tree.

The constructs of the invention can be introduced into plants by a variety of conventional transgenic techniques, such as by particle bombardment, by protoplast transformation by electroporation and microinjection, or by Agrobacterium-mediated transformation.

The beneficial effects of the invention include: the method can simply and conveniently select the plants with higher editing efficiency, can simply and conveniently obtain and screen out the plants without transgenic elements, and can also be used for multiple gene editing in plants.

In further embodiments of the invention, the invention relates to constructs, systems and methods for performing multiplex gene editing. In particular, the construct of the first aspect of the invention is used in combination with a pair of isocaudards, which are used for the assembly of multiple sgrnas.

"Cotailase", "Cotailrestriction enzyme" and "Cotailrestriction endonuclease" are used interchangeably in the context of the present invention and refer to a class of restriction endonucleases that recognize and cleave different target sequences but produce cohesive ends of identical sequence after cleavage. Thus, DNA fragments produced by digestion with two or more enzymes that are isocaudarner of each other can be ligated to each other by complementation between their cohesive ends. After DNA fragments cut by two different restriction enzymes with the same tail enzyme are connected through corresponding sticky ends, because the nucleotides of 5 'and 3' of a double-stranded sequence formed by the sticky end sequences are respectively from different restriction enzyme recognition sites, the corresponding positions of the connected fragments do not form the recognition site of any one of the two restriction enzymes which are originally cut and can not be recognized by the original restriction enzyme. Some sets of isocaudarner enzymes are listed below by way of example, each set including two or more restriction enzymes, with two isocaudarner enzymes in a set being isocaudarner with each other.

TABLE 1 examples of isocaudarner

In the present invention, expression cassettes of multiple sgrnas are ligated using a isocaudarner to form a construct useful for multiplex gene editing. The isocaudarner useful in the present invention may be selected from the group including but not limited to any of the following: EcoR I and Mfe I; pst I and Sbf I; any two of Xba I, Spe I and Nhe I; BamH I and Bgl II; and Xho I and Sal I. In a preferred embodiment of the invention, Spe I and Nhe I are used.

Examples

For a more complete understanding and appreciation of the invention, the invention will be described in detail below with reference to examples and the accompanying drawings, which are intended to illustrate the invention and not to limit the scope thereof. The scope of the invention is specifically defined by the appended claims.

Example 1 potency of 2A peptides from different sources

To obtain a CRISPR system that enables visual inspection of functional Cas9 abundance in plants, the present inventors explored a variety of self-cleaving peptides suitable for plants for linking Cas9 and a reporter protein. 2A peptides derived from picornavirus were demonstrated to exhibit self-cleaving properties in more than 100 animal cells and tissues (Kim et al, PLoS One 6, e18556,2011), and to test whether 2A peptides can also play a similar role in plants, and which 2A peptides have the best effect in plants, the following five different 2A peptides were tested: two 2A peptides derived from foot-and-mouth disease virus (F2A-1 and F2A-2, encoding nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:4, respectively), 2A peptide derived from equine influenza A virus (E2A, encoding nucleotide sequence of SEQ ID NO:3), 2A peptide derived from Toxoya Asia virus (Thosea asigna virus) (T2A, encoding nucleotide sequence of SEQ ID NO:6) and 2A peptide derived from porcine teschovirus (P2A, encoding nucleotide sequence of SEQ ID NO: 5). A peptide having a nucleotide sequence shown in SEQ ID NO:1 was also used as a Control Peptide (CP) which did not have the ability of the 2A peptide to undergo self-cleavage in vivo and was similar in length to the 2A peptide described above. The five 2A peptides and Control Peptides (CP) described above were used to link Cas9 and GFP, respectively, and then various Cas9-2A-GFP or Cas9-CP-GFP expression cassettes were ligated into constitutive expression vectors. Wherein two Nuclear Localization Signal (NLS) peptides are fused to the C-and N-termini of Cas9, respectively. The cleavage efficiency of these 2A peptides was assessed in protoplasts by confocal microscopy and western blot analysis. The experimental procedure will be described in detail below.

Plant material and growth conditions

In this example, the wild type Arabidopsis thaliana Columbia-0 type (Arabidopsis thaliana Columbia-0) was used. Seeds were sterilized with 15% NaClO for 10 min and then sterilized ddH₂And O washing for 5 times. Sterilized T1 seeds were grown on MS plates (4.4g/L Murashige Skoog powder, 1% sucrose, 8g/L agar, pH 5.8) containing 50mg/L hygromycin or 200g/L gentamicin sulfate.

T2 seeds were sown on MS plates without antibiotics. Placing the plate with seed at 4 deg.C for 4-5 days, placing the plate in 22 deg.C light incubator at 80 μmol · m^-2·s^-1Culturing under continuous white light. Transferring seven-day-old seedling to soil, and growing under long-day conditions (60-80 μmol. m)^-2·s^-1White light, 16h light 22 ℃/8h dark 18 ℃ cycle).

Plasmid construction

Constructs containing Cas9 and GFP fused with a 2A peptide were prepared for protoplast transformation as follows.

Cloning of GFP sequences

First, GFP was amplified from pJim19(Bar) -GFP (Sun et al, Proceedings of the National Academy of Sciences of the United States of America 113,6071-6076,2016), and cloned into pJim19(Gent) using Spe I and Sac I to generate pJim19(Gent) -GFP. The rbcS-E9 terminator was cloned from pHEE401E (Wang et al, Genome Biology 16,144,2015) and inserted between Sac I and EcoR I sites of pJim19(Gent) -GFP in place of the Nos terminator, yielding pJim19(Gent) -GFP-rbcS-E9 t.

Cloning of 2A peptides

The nucleotide sequences of the five 2A peptides and the Control Peptide (CP) described above, as well as the Kpn I and Spe I restriction enzyme sites, were synthesized, annealed and cloned into pJim19(Gent) -GFP-rbcS-E9t digested with Kpn I and Spe I to generate pJim19(Gent) -linker-GFP-rbcS-E9 t. Wherein the "linker" is any one of the nucleotide sequences of CP, F2A-1, E2A, F2A-2, P2A or T2A.

Cloning of Cas9

Cas9, which did not contain a stop codon, was then amplified from p35S-Cas9-SK (Feng et al, 2013, supra) and inserted into the pBS vector to generate pBS-Cas9 (which did not contain a stop codon).

Assembly of Cas9-2A-GFP expression cassette

The "linker-GFP-rbcS-E9 t" fragment was amplified from pJim19(Gent) -linker-GFP-rbcS-E9 t vector and cloned into pBS-Cas9 (without stop codon) to generate pBS-Cas 9-linker-GFP-rbcS-E9 t.

Finally, p35S-Cas9-SK was modified by deleting one Xho I site upstream of the dual CaMV35S promoter and adding a Nhe I site between Hind III and Sal I.

Both the modified p35S-Cas9-SK and pBS-Cas 9-linker-GFP-rbcS-E9 t were digested with Xho I and EcoR I to generate the final plasmid p35S-Cas 9-linker-GFP-rbcS-E9 t. See table 7 for all primer sequences.

sgRNA expression Module construction

To generate an expression module for multiple sgRNAs, the pAtU6-26-SK vector (Feng et al, 2013, supra) was modified by deleting the Spe I site downstream of the sgRNA Scaffold (Scaffold) and adding a Nhe I site between Xho I and Sal I to yieldpAtU6-26-M(FIG. 5 and SEQ ID NO:7 show the structure of the key part of the vector, i.e., the AtU6-26-Scaffold module used to construct multiple sgRNA concatemers). Multiple sgRNAs are respectively inserted into the BbsI site of pAtU6-26-M to obtain a sequence shown as SEQ ID NO: 39. Multiple sgRNA expression cassettes were assembled using Sal I and a pair of homologous tail restriction enzymes, Spe I/Nhe I, as shown in fig. 1D.

Construction of genome editing plasmid

To generate the final genome editing plasmid, multiple sgRNA expression modules assembled from the co-tailed restriction enzymes were digested with Kpn I and Sal I and then inserted into a binary vector containing Cas9-P2A-GFP as described above.

See table 7, following the examples, for all primer sequences.

Isolation and transfection of mesophyll protoplasts

Three to four weeks old Arabidopsis thaliana were used for protoplast isolation by the procedure described below (Yoo et al, Nature Protocol 2,1565-1572, 2007). The leaves were cut into 0.5-1mM strips and immediately placed in 15ml of enzyme solution (1.87% cellulase R10, 0.32% macerase R10, 0.4M mannitol, 20mM MES, pH 5.7, 20mM KCl, 10mM CaCl)₂5mM beta-mercaptoethanol and 0.1% BSA) for 3 hours. Digested product was washed with 15mL of pre-cooled W5 solution (154mM NaCl, 125mM CaCl)₂5mM KCl and 2mM MES, pH 5.7) and filtered through a 70 μm nylon mesh. The protoplasts were pelleted by centrifugation at 1400rpm for 2 minutes and washed with 10ml of W5 solution, then centrifuged again. The protoplasts were resuspended using 10mL of pre-cooled W5 buffer, incubated on ice for 30 minutes, and the supernatant was discarded. MMG buffer (0.4M mannitol, 15mM MgCl)₂And 4mM MES, pH 5.7) was added to the protoplasts so that the concentration of the protoplasts was 10⁵-10⁶cells/mL. DNA transfection was performed using a 2mL round bottom microcentrifuge tube. Mu.l protoplasts were mixed with 20. mu.g plasmid and 220. mu.l PEG solution (40% PEG 4000, 0.2M mannitol and 0.1M CaCl₂) And (4) fully mixing. After 5 min incubation at room temperature, the transformation reaction was quenched by addition of 1mL of pre-cooled W5 buffer and the transfected protoplasts were collected by centrifugation at 1200rpm for 2 min. Next, the transfected protoplasts were resuspended in 100. mu. l W5 solution and transferred to a six-well plate precoated with 1ml of WI solution (0.5M mannitol, 20mM KCl, 5% fetal bovine serum and 4mM MES, pH 5.7). The plates were covered with toilet paper and placed in an incubator for 16 hours, and then total protein was observed or extracted under a microscope.

Protein extraction and western blotting

For theProtoplasts, the cultured protoplasts were collected by centrifugation at 100g for 1 minute. Add 20. mu.l of denaturation buffer (8M Urea, 100mM NaH)₂PO₄100mM Tris-HCl, pH 8.0, 1mM PMSF and 1 XProteinase inhibitor) and 5. mu.l of 5 XPSDS loading buffer (50% glycerol, 10% SDS, 0.1% bromophenol blue, 10mM DTT, 5% beta-mercaptoethanol and 250mM Tris-HCl, pH 6.8). The well mixed protoplasts and buffer were boiled at 100 ℃ for 10 minutes.

For plants, light-grown (light-grow) seedlings of 7 days old were harvested and ground to a powder in liquid nitrogen. Total protein was extracted using denaturing buffer. After centrifugation at 13,000rpm for 10 minutes, the supernatant containing total protein was mixed with 5x SDS loading buffer and then boiled at 100 ℃ for 10 minutes.

Protein samples were separated on 8% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% milk, the membranes were incubated with anti-GFP, anti-RPN 6 or anti-RPT 5 antibodies. Thereafter, the membrane was washed 3 times and incubated with the corresponding secondary antibody, washed 3 more times, and then detected with ECL primers (GE Healthcare).

Results

As can be seen in fig. 1A, unlike the case where the fluorescence was localized in the nucleus when transformed with Cas9-CP-GFP, some of the GFP fluorescence in protoplasts transformed with Cas9-2A-GFP was clearly localized in the cytoplasm, indicating that the GFP protein had been successfully cleaved from Cas 9. Furthermore, among all these 2A peptides, P2A showed the highest cleavage efficiency, which showed a self-cleavage efficiency of about 95% in protoplasts (see fig. 1B and 1C). Since Cas9 is correlated with GFP expression levels, and by efficient self-cleavage of P2A, GFP fluorescence intensity can well reflect Cas9 abundance, while GFP does not disrupt Cas9 function. Therefore, P2A was selected to fuse Cas9 and GFP for subsequent experiments.

Example 2 Effect of different promoters

The promoter is one of the key factors in CRISPR systems that can affect editing efficiency. Some previous studies have shown that the Cas9 system regulated by the 35S promoter is not efficient and the induced somatic mutations cannot be inherited.

To select an efficient promoter driving Cas9-P2A-GFP and test whether GFP strength is related to gene editing efficiency, the arabidopsis BRI1 (brassicasteroid-Insensitive 1) gene was selected as the target gene to study different promoters, including the dual CaMV35S promoter and the arabidopsis ubiquitin 10(UBQ10) gene promoter, and to test different vectors, including pJim19 and pCAMBIA 1300.

The BRI1 gene encodes a receptor protein with kinase activity that regulates the signaling cascade of growth and development through binding to brassinosteroids. If the BRI1 gene is edited to function improperly, the edited plant will have a severely stunted phenotype and become sterile.

To test the efficacy of both promoters, four different constructs were co-generated using two vectors, and constructed in the following manner.

The binary vector pJim19(Gent) (SEQ ID NO:40) was modified with sequences for Hind III, Stu I, Spe I, Kpn I, and EcoR I to replace the CaMV35S promoter and Nos terminator to generate pJim19(Gent) -M vector. The UBQ10 promoter was cloned from Arabidopsis genomic DNA using UBQ10NheI F and UBQ10XhoI R primers. UBQ10 promoter and P35S Cas9-P2A-GFP-rbcS-E9t vector were digested with Nhe I and Xho I, and assembled to give pUBQ10-Cas9-P2A-GFP-rbcS-E9t vector. UBQ10-Cas9-P2A-GFP-rbcS-E9t was cloned into EcoR I and Kpn I digested pJim19(Gent) -M to generate binary vector pJim19-ProUBQ10, Cas9-P2A-GFP-rbcS-E9t, abbreviated aspUBQ10:Cas9-P2A-GFP(Gent)。

In a similar manner the 2X 35S-Cas9-P2A-GFP-rbcS-E9t module was digested from P35S-Cas9-P2A-GFP-rbcS-E9t and cloned into pJim19(Gent) -M to obtain the binary vector pJim 19-2X 35S-Cas9-P2A-GFP-rbcS-E9t, abbreviated as pJim 19-2X 35S-Cas 9-GFP-rbcS-E9 tp35S:Cas9-P2A-GFP(Gent)。

In addition, the Cas9-P2A-GFP expression cassette was inserted into a modified version of pCAMBIA1300-M (SEQ ID NO:41) having hygromycin resistance to generate two additional binary vectorspUBQ10:Cas9-P2A-GFP(Hyg)Andp35S:Cas9-P2A-GFP(Hyg)。

since dual CaMV35S promoter is often used to regulate the expression of Cas9, the BRI1sgRNA expression cassette is first cloned into a Cas9-P2A-GFP carrying binary vector P35S: Cas9-P2A-GFP (gent) driven by dual CaMV35S promoter, which is then delivered into wild type by Agrobacterium (Agrobacterium). Of the 60T 1 transgenic plants, 5 seedlings with strong GFP fluorescence all showed severe defects in growth and a sterile phenotype, three of which are shown in FIG. 3A. In contrast, only 3 of the T1 plants that did not exhibit significant GFP fluorescence exhibited a semi-dwarf phenotype (see table 2). It was further confirmed by Sanger sequencing that all 5 lines with high GFP expression edited the target locus and resulted in chimeric mutations (as in figure 3B and table 2).

Then, the BRI1sgRNA expression cassette was inserted into a binary vector pUBQ10: Cas9-P2A-GFP (Gent) with Cas9-P2A-GFP driven by the Arabidopsis ubiquitin 10(AtUBQ10) gene promoter. Of the 47T 1 transgenic seedlings, 10 plants showed strong GFP fluorescence and all showed a severe dwarf phenotype similar to the bri1 mutant when grown in soil (see figure 3C and table 2). Sequencing data showed that 3 of 10 seedlings were homozygous mutants (fig. 3D and table 2). Unexpectedly, 12T 1 seedlings, which did not have significant GFP fluorescence, also exhibited a severely stunted phenotype, with both strains being homozygous mutants. These results indicate that the editing efficiency of T1 transgenic plants with high GFP fluorescence was higher for both CaMV35S promoter and UBQ10 promoter than for T1 transgenic plants without significant fluorescence (table 2). The results also indicate that UBQ10 is superior to the 35S promoter in driving Cas9-P2A-GFP editing of the genome.

TABLE 2 summary of phenotype and genotype of T1 generation plants edited on pJim19 vector using dual CaMV35S promoter (2X35S) or ubiquitin 10 promoter (UBQ10)

Example 3 multiplex editing Using isocaudarner

To explore whether the system of the invention is suitable for multiplex gene editing, the construct of the invention was combined with a tailgating restriction enzyme-based assembly method. Since the experiment in this example was performed before the experiment in example 2, the 35S promoter was still used.

To generate an expression cassette for multiple sgRNAs, the pAtU6-26-SK vector (Feng et al, 2013, supra) was modified by deleting the Spe I site downstream of the sgRNA scaffold (scaffold) and adding a Nhe I site between Xho I and Sal I to yieldpAtU6-26-M(FIG. 5).

Multiple sgrnas were inserted into the BbsI site of pAtU 6-26-M. Multiple sgRNA expression cassettes were assembled using Sal I and a pair of homologous tail restriction enzymes, Spe I/Nhe I, as shown in fig. 1D.

To generate the final genome editing plasmid, multiple sgRNA expression cassettes assembled from the co-tailed restriction enzymes were digested with Kpn I and Sal I and then inserted into a binary vector containing Cas9-P2A-GFP as described above.

Primers for the sgrnas were annealed out and inserted AtU6-26 between the scaffold via the Bbs I site (fig. 4 and 5). As shown in FIG. 4, the method of this example utilizes a pair of isocaudarner Spe I and Nhe I, which produce the same sticky-end CTAG after cleaving double-stranded DNA. After digestion and ligation using Spe I/Sal I and Nhe I/Sal I, multiple sgRNA expression cassettes were assembled together in series (FIG. 1D). Then, an expression cassette containing multiple tandem sgrnas was digested with Kpn I and Sal I and inserted into a binary vector containing Cas9-P2A-GFP driven by the dual CaMV35S promoter or the arabidopsis thaliana ubiquitin 10 gene (AtUBQ10) promoter as constructed in examples 1 and 2 (fig. 1E; fig. 4 and 2). Next, plants were transformed with these constructs, transformed seeds were grown on antibiotic-containing MS plates, and seedlings with high GFP expression were selected by fluorescence stereoscopy or microscopy. T1 transgenic seedlings with strong GFP fluorescence were transferred to soil to produce progeny. Finally, individuals not expressing GFP were selected in transgenic plants of the T2 generation and genotyped, and mutants not carrying transgenic elements and edited from multiple genes were finally obtained (see fig. 3A). Using this multiple editing construct of the invention, protein levels of Cas9 were easily detected by examining fluorescence intensity in transgenic plants, while achieving editing of multiple genes. By selecting T1 plants with high GFP fluorescence and T2 plants without fluorescence, a multiplex mutant without Cas9 can be obtained easily. The specific results are described in example 4.

Example 4 selection of multiple mutants not carrying a transgenic element based on simple fluorescence detection

The system of the invention can utilize Cas9-P2A-GFP constructs to generate plants that are subject to multiple gene edits and that do not contain a transgenic element.

First, multiple sgRNA expression elements were assembled using a co-tailed restriction endonuclease, and the assembled multiple sgRNA expression cassettes were inserted into a binary vector containing Cas9-P2A-GFP using Kpn I and Sal I (as described in example 3, see fig. 4). Among the T2 transgenic plants, plants without GFP expression were selected and genotyped to obtain mutants carrying no transgenic elements and undergoing multiple gene editing (scheme shown in FIG. 2A).

To test the efficiency of the CRISPR system of the invention, BZR1 and BES1 were selected as targets for generating mutants that do not carry transgenic elements. Four sgrnas (as shown in fig. 6B, primers see table 7), two for BZR1 and two for BES1 were designed and inserted into the P35S: Cas9-P2A-gfp (hyg) binary vector constructed as in example 2. Wild type (Col-0) plants were transformed. 4 seedlings with high GFP expression were selected from 62T 1 plants on a wild-type background, the fluorescence of 2 representative seedlings of which is shown in FIG. 6A, and the number of plants with fluorescence in the T1 plants, and the number of mutations contained in these fluorescent plants, are summarized in Table 3 below.

TABLE 3 fluorescence and Gene editing efficiency in T1 plants

In representative seedlings without apparent GFP expression, all four target sites were unedited (as in fig. 6B), but efficiently edited in plants with high GFP expression (fig. 6C and 6D). In the T2 progeny of these two representative T1 plants with high GFP expression, the segregation ratio of GFP and non-GFP plants was close to 3:1, indicating a single transgene insertion (fig. 2B and table 7).

TABLE 4 fluorescence and Gene editing efficiency in T2 plants

Most of the Cas9-P2A-GFP protein in the fluorescent T2 plants was self-cleaved (fig. 7A), indicating that P2A works well in arabidopsis plants, consistent with the results in arabidopsis protoplasts (fig. 1A to 1C).

Further genotyping of T2 plants was performed using Cas 9-specific primers, which confirmed the perfect correspondence between GFP fluorescence and Cas 9-containing DNA fragments (fig. 2C; fig. 7B). Sanger sequencing showed that all four sgRNA loci were efficiently edited (see table 5 below).

TABLE 5 editing efficiency of four sgRNAs in T2 generation plants not carrying the transgenic element

The inventors surprisingly found that 8/40 (20%) T2 plants of line #10 had mutations at all four loci, while 3/40 (7.5%) T2 plants were homozygous at all four loci (table 6). Sequencing results for representative homozygous lines are shown in fig. 2D.

TABLE 6 Simultaneous editing of four loci T2 generation plants not carrying a transgene element

Underlined indicates that all four target sites are homozygous mutations

In another study, the inventors constructed a module containing 12 sgRNA expression cassettes together to target 8 SAUR genes using the method shown in fig. 1D, and found that at least 10 sgrnas functioned normally.

These results indicate that the novel CRISPR system of the invention using Cas9-P2A-GFP conveniently and efficiently generates multiple mutants without carrying a transgenic element.

The above results also demonstrate the importance of high expression of Cas9 for efficient editing, particularly in the case of multiple editing using multiple sgrnas, as multiple sgrnas may compete for Cas9 protein. These results also demonstrate that the CRISPR system of the invention using Cas9-P2A-GFP enables the screening of plants highly expressing Cas9 to be simple and easy to implement. Previous methods, whether they do not screen for T1 generation plants or screen for genotypes, are not as efficient as the method of the present invention. By using the construct and the method, T1 generation plants selected by the marker with strong fluorescence have higher Cas9 expression, and T2 generation plants which do not carry CRISPR/Cas9 transgenic elements can be obtained by higher proportion of the plants, so that the efficiency of generating ideal multi-editing mutants which do not carry CRISPR/Cas9 transgenic elements is improved, and the workload is greatly reduced.

The above examples demonstrate the efficacy of the system of the invention in the model plant Arabidopsis thaliana, and also demonstrate its utility in other plants.

TABLE 7 primers used in the examples

Sequence listing

<110> Beijing university

<120> CRISPR/Cas9 system and method for efficient generation of mutants not carrying a transgenic element in plants

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<170> SIPOSequenceListing 1.0

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<213> Foot-and-mouth disease Virus (Foot-and-mouth disease virus)

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ggacct 66

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ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 60

cct 63

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ggtaccgagc tcggatccac tagtaacggc cgccagtgtg ctggaattgc ccttaagctt 60

cgttgaacaa cggaaactcg acttgccttc cgcacaatac atcatttctt cttagctttt 120

tttcttcttc ttcgttcata cagttttttt ttgtttatca gcttacattt tcttgaaccg 180

tagctttcgt tttcttcttt ttaactttcc attcggagtt tttgtatctt gtttcatagt 240

ttgtcccagg attagaatga ttaggcatcg aaccttcaag aatttgattg aataaaacat 300

cttcattctt aagatatgaa gataatcttc aaaaggcccc tgggaatctg aaagaagaga 360

agcaggccca tttatatggg aaagaacaat agtatttctt atataggccc atttaagttg 420

aaaacaatct tcaaaagtcc cacatcgctt agataagaaa acgaagctga gtttatatac 480

agctagagtc gaagtagtga ttgggtcttc gagaagacct gttttagagc tagaaatagc 540

aagttaaaat aaggctagcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 600

ttgtcccttc gaagggcctt tctcagatat ccatcacact ggcggccgct cgaggctagc 660

gatatcgtcg acggtatcga taagcttgat atc 693

<210> 8

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<223> BRI1 sgRNA R

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aaactcggta tgtatccaga caac 24

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<223> BRI1-Cas9 test F

<400> 10

agaaagagtg tcatggagct ggt 23

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tcccatcgag cttattgctt g 21

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<400> 12

gattgagaaa gggagaataa tcgg 24

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1 sgRNA1 R

<400> 13

aaacccgatt attctccctt tctc 24

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<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1 sgRNA2 F

<400> 14

gattggtgca gaaaccgcat agaa 24

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1 sgRNA2 R

<400> 15

aaacttctat gcggtttctg cacc 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1 sgRNA1 F

<400> 16

gattgcggcg gagaagagct gttg 24

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1 sgRNA1 R

<400> 17

aaaccaacag ctcttctccg ccgc 24

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1 sgRNA2 F

<400> 18

gattgaagtt ggggatgaca ctgg 24

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1 sgRNA2 R

<400> 19

aaacccagtg tcatccccaa cttc 24

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1-Cas9 test F1

<400> 20

cgtgagcact aacttctcac tcctc 25

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1-Cas9 test R1

<400> 21

ccctcaaatt ctcagattca aacc 24

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1-Cas9 test F2

<400> 22

gttgtccagt taccccaccg gtc 23

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BZR1-Cas9 test R2

<400> 23

gaaggcagca gtatttggag acatc 25

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1-Cas9 test F1

<400> 24

gagagttgaa ggaagaagat gacg 24

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1-Cas9 test R1

<400> 25

atgcgtttga tactgacctt gcg 23

<210> 26

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1-Cas9 test F2

<400> 26

cccataacca aagtcctctt tcttcc 26

<210> 27

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> BES1-Cas9 test R2

<400> 27

ctcttggatt gctgctgtgt ttggag 26

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Cas9 T-DNA test F

<400> 28

gtggaccata tcgtgcctca gag 23

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Cas9 T-DNA test R

<400> 29

ctgcacctcg gtctttttca cg 22

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCambia1300 Modified F

<400> 30

aattccgggg taccgctcta gat 23

<210> 31

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCambia1300 Modified R

<400> 31

agctatctag agcggtaccc cgg 23

<210> 32

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> UBQ10 NheI F

<400> 32

ctagctagcg accgttagaa attgtggttg tcgaggagtc agtaataaac ggcgtcaaa 59

<210> 33

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> UBQ10 XhoI R

<400> 33

ccgctcgagc tgttaatcag aaaaactcag attaatcg 38

<210> 34

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AtU6 delet SpeI F

<400> 34

aattcctgca gcccgggt 18

<210> 35

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AtU6 delet SpeI R

<400> 35

ctagacccgg gctgcagg 18

<210> 36

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AtU6 add NheI F

<400> 36

tcgaggctag cgatatcg 18

<210> 37

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AtU6 add NheI R

<400> 37

tcgacgatat cgctagcc 18

<210> 38

<211> 656

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> UBQ10 promoter

<400> 38

gaccgttaga aattgtggtt gtcgaggagt cagtaataaa cggcgtcaaa gtggttgcag 60

ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa tacttttcct caacctaaaa 120

ataaggcaat tagccaaaaa caactttgcg tgtaaacaac gctcaataca cgtgtcattt 180

tattattagc tattgcttca ccgccttagc tttctcgtga cctagtcgtc ctcgtctttt 240

cttcttcttc ttctataaaa caatacccaa agagctcttc ttcttcacaa ttcagatttc 300

aatttctcaa aatcttaaaa actttctctc aattctctct accgtgatca aggtaaattt 360

ctgtgttcct tattctctca aaatcttcga ttttgttttc gttcgatccc aatttcgtat 420

atgttctttg gtttagattc tgttaatctt agatcgaaga cgattttctg ggtttgatcg 480

ttagatatca tcttaattct cgattagggt ttcatagata tcatccgatt tgttcaaata 540

atttgagttt tgtcgaataa ttactcttcg atttgtgatt tctatctaga tctggtgtta 600

gtttctagtt tgtgcgatcg aatttgtcga ttaatctgag tttttctgat taacag 656

<210> 39

<211> 3592

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pAtU6-26-M-sgRNA construct

<220>

<222> (707)..(1255)

<223> AtU6-sgRNA

<400> 39

cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 60

tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 120

tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 180

gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 240

gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 300

atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 360

atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 420

aatttaacgc gaattttaac aaaatattaa cgcttacaat ttccattcgc cattcaggct 480

gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 540

agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 600

ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccga 660

gctcggatcc actagtaacg gccgccagtg tgctggaatt gcccttaagc ttcgttgaac 720

aacggaaact cgacttgcct tccgcacaat acatcatttc ttcttagctt tttttcttct 780

tcttcgttca tacagttttt ttttgtttat cagcttacat tttcttgaac cgtagctttc 840

gttttcttct ttttaacttt ccattcggag tttttgtatc ttgtttcata gtttgtccca 900

ggattagaat gattaggcat cgaaccttca agaatttgat tgaataaaac atcttcattc 960

ttaagatatg aagataatct tcaaaaggcc cctgggaatc tgaaagaaga gaagcaggcc 1020

catttatatg ggaaagaaca atagtatttc ttatataggc ccatttaagt tgaaaacaat 1080

cttcaaaagt cccacatcgc ttagataaga aaacgaagct gagtttatat acagctagag 1140

tcgaagtagt gattgggtct tcgagaagac ctgttttaga gctagaaata gcaagttaaa 1200

ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgtccc 1260

ttcgaagggc ctttctcaga tatccatcac actggcggcc gctcgaggct agcgatatcg 1320

tcgacggtat cgataagctt gatatcgaat tcctgcagcc cgggtctaga gcggccgcca 1380

ccgcggtgga gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa 1440

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 1500

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1560

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1620

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1680

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1740

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1800

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1860

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1920

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1980

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2040

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2100

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2160

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 2220

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2280

actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2340

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 2400

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 2460

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 2520

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2580

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 2640

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 2700

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 2760

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 2820

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 2880

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 2940

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3000

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3060

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3120

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 3180

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 3240

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 3300

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 3360

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3420

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 3480

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 3540

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gc 3592

<210> 40

<211> 8388

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pJim19 (Gent)-M

<220>

<222> (5023)..(5043)

<223> pJim19 Genta M seq R

<220>

<222> (5132)..(5161)

<223> restriction enzyme recognition site

<220>

<222> (5280)..(5301)

<223> pJim19 Genta M seq F

<400> 40

gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg 60

ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag 120

ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga 180

tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga 240

cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg 300

cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca 360

gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa 420

atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca 480

tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga 540

tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata 600

gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc 660

caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag 720

gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct 780

gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt 840

cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa 900

aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac 960

tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa 1020

aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 1080

agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 1140

acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 1200

ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 1260

accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 1320

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 1380

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 1440

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 1500

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 1560

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 1620

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 1680

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 1740

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 1800

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 1860

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 1920

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 1980

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 2040

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 2100

gttaagggat tttggtcatg catgatatat ctcccaattt gtgtagggct tattatgcac 2160

gcttaaaaat aataaaagca gacttgacct gatagtttgg ctgtgagcaa ttatgtgctt 2220

agtgcatcta acgcttgagt taagccgcgc cgcgaagcgg cgtcggcttg aacgaatttc 2280

tagctagaca ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt 2340

cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc 2400

tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag 2460

cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa 2520

gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt 2580

catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg 2640

gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt 2700

cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat tctccaaatt 2760

gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga 2820

cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt 2880

tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa 2940

tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca 3000

acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt 3060

cggcgatcac cgcttccccc atgatgttta actttgtttt agggcgactg ccctgctgcg 3120

taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct 3180

tggatgcccg aggcatagac tgtaccccaa aaaaacagtc ataacaagcc atgaaaaccg 3240

ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca 3300

tacgctactt gcattacagc ttacgaaccg aacaggctta tgtccactgg gttcgtgccc 3360

gaattgatca caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga 3420

gatcatccgt gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac 3480

atgagcaaag tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg 3540

ctgcctgtat cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct 3600

ggtggcagga tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg 3660

cggacgtttt taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta 3720

ctggattttg gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat 3780

acatactaag ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt 3840

cccttatctg ggaactactc acacattatt atggagaaac tcgagcttgt cgatcgactc 3900

taggccggga agccgatctc ggcttgaacg aattgttagg tggcggtact tgggtcgata 3960

tcaaagtgca tcacttcttc ccgtatgccc aactttgtat agagagccac tgcgggatcg 4020

tcaccgtaat ctgcttgcac gtagatcaca taagcaccaa gcgcgttggc ctcatgcttg 4080

aggagattga tgagcgcggt ggcaatgccc tgcctccggt gctcgccgga gactgcgaga 4140

tcatagatat agatctcact acgcggctgc tcaaacttgg gcagaacgta agccgcgaga 4200

gcgccaacaa ccgcttcttg gtcgaaggca gcaagcgcga tgaatgtctt actacggagc 4260

aagttcccga ggtaatcgga gtccggctga tgttgggagt aggtggctac gtctccgaac 4320

tcacgaccga aaagatcaag agcagcccgc atggatttga cttggtcagg gccgagccta 4380

catgtgcgaa tgatgcccat acttgagcca cctaactttg ttttagggcg actgccctgc 4440

tgcgtaacat cgttgctgct gcgtaccatg gtcgatcgac agatctgcga aagctcgaga 4500

gagatagatt tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac 4560

ttccttatat agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt 4620

cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 4680

ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 4740

gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta 4800

ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata 4860

ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat 4920

tcttggagta gacgagagtg tcgtgctcca ccatgtttgc aagctgctct agcattcgcc 4980

attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 5040

gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 5100

gtcacgacgt tgtaaaacga cggccagtgc caagcttagg cctactagtg gtaccgaatt 5160

cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 5220

catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 5280

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 5340

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attggctaga gcgctctaga 5400

gcagcttgag cttggatcag attgtcgttt cccgccttca gtttaaacta tcagtgtttg 5460

acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat aatcggatat 5520

ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg 5580

gttcccctcg ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct 5640

tctgacgttc agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc taagttacgc 5700

gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa 5760

agtagaatac ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg 5820

gcctgctggg ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg 5880

aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg 5940

accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga 6000

ccaggctaga ccgcctggcc cgcagcaccc gcgacctact ggacattgcc gagcgcatcc 6060

aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc accacgccgg 6120

ccggccgcat ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt tccctaatca 6180

tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc 6240

gccctaccct caccccggca cagatcgcgc acgcccgcga gctgatcgac caggaaggcc 6300

gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac 6360

ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg 6420

acgcattgac cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag 6480

catgaaaccg caccaggacg gccaggacga accgtttttc attaccgaag agatcgaggc 6540

ggagatgatc gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct 6600

gcatgaaatc ctggccggtt tgtctgatgc caagctggcg gcctggccgg ccagcttggc 6660

cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt 6720

gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac gcaaggggaa 6780

cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt caggcaagac gaccatcgca 6840

acccatctag cccgcgccct gcaactcgcc ggggccgatg ttctgttagt cgattccgat 6900

ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc 6960

ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta 7020

gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac 7080

ttcgtgctga ttccggtgca gccaagccct tacgacatat gggccaccgc cgacctggtg 7140

gagctggtta agcagcgcat tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg 7200

tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac 7260

gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg cactgccgcc 7320

gccggcacaa ccgttcttga atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg 7380

ctggccgctg aaattaaatc aaaactcatt tgagttaatg aggtaaagag aaaatgagca 7440

aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt 7500

tggccagcct ggcagacacg ccagccatga agcgggtcaa ctttcagttg ccggcggagg 7560

atcacaccaa gctgaagatg tacgcggtac gccaaggcaa gaccattacc gagctgctat 7620

ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag tagatgaatt 7680

ttagcggcta aaggaggcgg catggaaaat caagaacaac caggcaccga cgccgtggaa 7740

tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg 7800

ccctgcaatg gcactggaac ccccaagccc gaggaatcgg cgtgagcggt cgcaaaccat 7860

ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg 7920

cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag 7980

cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga 8040

ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg ctctatgacg 8100

tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg 8160

accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta gaggtttccg 8220

cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg gcggtttccc 8280

atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc ggccgcgtgt 8340

tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatg 8388

<210> 41

<211> 8930

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pCAMBIA1300-M

<400> 41

gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 60

taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 120

aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 180

atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 240

tacgaattcc ggggtaccgc tctagatagc ttggcactgg ccgtcgtttt acaacgtcgt 300

gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 360

agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 420

aatggcgaat gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt 480

aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat 540

tagaataacg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg 600

catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 660

atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 720

agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 780

gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 840

agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 900

ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 960

ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 1020

acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 1080

ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 1140

acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 1200

agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 1260

tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 1320

tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 1380

ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 1440

gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 1500

gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 1560

cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1620

ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1680

gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1740

tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1800

aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1860

aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1920

ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1980

ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 2040

cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 2100

atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 2160

accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 2220

gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 2280

gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 2340

ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 2400

cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 2460

aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 2520

gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 2580

agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2640

ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2700

atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2760

accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2820

tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2880

cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2940

gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 3000

tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 3060

cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 3120

gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 3180

tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 3240

cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 3300

gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 3360

gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 3420

tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 3480

tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 3540

agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3600

gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3660

gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3720

ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3780

cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3840

aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3900

catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3960

gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 4020

tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 4080

cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 4140

aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 4200

ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 4260

gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 4320

aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 4380

actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 4440

aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 4500

ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 4560

tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4620

gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4680

ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4740

gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4800

ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4860

ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4920

acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4980

tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 5040

ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 5100

ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 5160

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 5220

actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5280

gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 5340

tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5400

caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5460

atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5520

acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 5580

atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5640

ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5700

gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5760

gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5820

ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5880

gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5940

taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 6000

cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 6060

gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 6120

gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 6180

atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 6240

tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 6300

tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 6360

tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 6420

aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 6480

ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 6540

caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6600

gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6660

tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6720

cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6780

tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6840

taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6900

gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6960

ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 7020

ggaactactc acacattatt atggagaaac tcgagcttgt cgatcgacag atccggtcgg 7080

catctactct atttctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg 7140

agtacttcta cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc 7200

ccgacagtcc cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca 7260

tcatcgaaat tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata 7320

tacgcccgga gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc 7380

tgctgctcca tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg 7440

gaatccccga acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc 7500

aggacattgt tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg 7560

gcccaaagca tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc 7620

acagtttgcc agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta 7680

gtgtattgac cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg 7740

gccgcagcga tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt 7800

tcaggcaggt cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc 7860

tcgctaaact ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc 7920

cgataaacat aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat 7980

ccacgccctc ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc 8040

aggtcggaga cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt 8100

tcaggctttt tcatatctca ttgccccccg ggatctgcga aagctcgaga gagatagatt 8160

tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat 8220

agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata 8280

tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 8340

ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 8400

tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 8460

atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 8520

tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 8580

acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt 8640

ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 8700

tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 8760

ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 8820

aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 8880

actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat 8930

Claims (12)

1. A construct of a CRISPR/Cas9 gene editing system for plants comprising a Cas9 expression cassette, said Cas9 expression cassette comprising, from 5 'to 3':

(a) the Arabidopsis ubiquitin 10 gene (AtUBQ10) promoter for the Cas9 expression cassette,

(b) a nuclease coding sequence which is capable of coding a nuclease,

(c) self-cleaving peptide coding sequences, and

(d) a sequence of a reporter gene,

wherein (a) and (b) - (d) are operably linked such that the nuclease coding sequence and reporter sequence are under the control of the promoter, the nuclease coding sequence encodes Cas9 or a fragment or variant thereof having DNA cleavage activity, and the self-cleaving peptide coding sequence encodes a 2A peptide derived from porcine teschovirus and is the nucleotide sequence set forth in SEQ ID NO:5 or a mutant thereof encoding the same polypeptide.

2. The construct of claim 1, wherein the reporter gene encodes a protein selected from the group consisting of: luciferase, green fluorescent protein, red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein.

3. The construct of claim 1, wherein the promoter is a promoter constitutively expressed in a plant.

4. The construct of claim 1, wherein the construct comprises a Nuclear Localization Signal (NLS) coding sequence flanking both sides of the nuclease coding sequence.

5. The construct of any one of claims 1-4, further comprising a sgRNA module comprising one or more sgRNA expression cassettes, wherein each sgRNA expression cassette comprises, from 5 'to 3':

(e) a promoter for the sgRNA expression cassette; and

(f) sgRNA for the target DNA sequence to be edited.

6. A method of making a construct for CRISPR/Cas9 gene editing in a plant comprising introducing into a vector a Cas9 expression cassette, said Cas9 expression cassette comprising, from 5 'to 3':

(a) the Arabidopsis ubiquitin 10 gene (AtUBQ10) promoter for the Cas9 expression cassette,

(b) a nuclease coding sequence which is capable of coding a nuclease,

(c) self-cleaving peptide coding sequences, and

(d) a sequence of a reporter gene,

wherein (a) and (b) - (d) are operably linked such that the nuclease coding sequence and reporter sequence are under the control of the promoter, the nuclease coding sequence encodes Cas9 or a fragment or variant thereof having DNA cleavage activity, and the self-cleaving peptide coding sequence encodes a 2A peptide derived from porcine teschovirus and is the nucleotide sequence set forth in SEQ ID NO:5 or a mutant thereof encoding the same polypeptide.

7. The method of claim 6, further comprising introducing into the vector a sgRNA module comprising one or more sgRNA expression cassettes, wherein each sgRNA expression cassette comprises, from 5 'to 3':

(e) a promoter for the sgRNA expression cassette; and

(f) sgRNA against a labeled target DNA sequence.

8. The method of claim 7, wherein the sgRNA module comprising a plurality of sgRNA expression cassettes is obtained by assembling together a plurality of sgRNA expression cassettes, the assembling comprising adding upstream and downstream restriction enzymes, respectively, upstream and downstream of each sgRNA expression cassette, the upstream and downstream restriction enzymes being different restriction enzymes and being tailzymes of each other, wherein the upstream restriction enzyme added for all sgRNA expression cassettes is the same and the downstream restriction enzyme added for all sgRNA expression cassettes is the same.

9. Construct obtained by the method of any one of claims 6 to 8.

10. A method of gene editing in a plant comprising the use of a construct according to any one of claims 1 to 5 or 9.

11. A method for obtaining a mutant not carrying a transgenic element in a plant, comprising:

(i) transforming a plant with the construct of claim 5 or 9;

(ii) selecting a first generation plant with positive reporter gene expression as a transformant;

(iii) (iii) passaging the first generation plants in (ii) to obtain second generation plants; and

(iv) second generation plants negative for reporter gene expression were selected as mutants that were gene edited and did not carry transgenic elements.

12. The method of claim 11, wherein after selecting a plant by reporter gene expression in steps (ii) and (iv), identifying the target gene sequence of the selected plant to confirm that it is edited.

Images