CN114540406B - Genome editing expression frame, vector and application thereof - Google Patents

Abstract

The invention belongs to the field of plant gene editing, and particularly relates to a genome editing expression frame, a vector containing the expression frame and application thereof. The technical problem to be solved by the invention is that a high-efficiency gene editing system which is not applicable to plants, particularly gymnosperms at present. The technical scheme for solving the technical problem is to provide a genome editing expression frame, which comprises the following elements: the promoter LarPE004, the encoding gene of the Cas9 protein, the sgRNA cloning and transcription unit are used for driving the expression of the encoding gene of the Cas9 protein and the sgRNA cloning and transcription unit by the promoter LarPE 004. The expression frame and the corresponding vector can realize efficient gene editing in plants. Experiments show that the gene editing efficiency in larch protoplast can reach 72.5%, and simultaneously, the gene editing activity in a plurality of PAM sites such as PAM NGG, NAG, NGT is higher, thus having wide application prospect.

Description

Genome editing expression frame, vector and application thereof

Technical Field

The invention belongs to the field of plant gene editing, and particularly relates to a genome editing expression frame, a vector containing the expression frame and application thereof.

Background

The plant genome editing technique (PlantGenome Editing Technology) is to artificially edit the plant genome DNA by using a genome editing tool, and introduce a mutation form such as a predetermined base insertion, deletion, substitution, etc., so that the plant genome DNA is changed in a targeted manner according to the human will. Plant genome editing tools have been explored and evolved over ten years, and third generation tools for plant genome editing using clustered regularly interspaced short palindromic repeat-effector nuclease (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated Cas, CRISPR-Cas) systems have been invented. The CRISPR-Cas genome editing tool has the advantages of low experimental cost, easy vector construction, high detection speed, strong developability and the like, and is the most widely used tool in plant genome editing work at present. Researchers have successfully applied CRISPR-Cas systems in a variety of model plants, such as tobacco, arabidopsis; in various crops such as wheat, corn, rice, sorghum; among a variety of woody plants, such as apples, grapes, citrus; a genome editing platform comprising gene knockout, single-base editing and accurate gene insertion is built in various herbaceous plants such as cucumbers, potatoes and tomatoes, great progress is made, and high editing efficiency is achieved for part of gene loci in model plants such as arabidopsis, rice and the like.

Larch (Larix kaempferi) is a cold-temperate and temperate larry arbor, has the characteristics of rapid growth in early stage, strong stress resistance and disease resistance, wide adaptability, strong conservation of water sources and good ecological benefit, is a very important afforestation tree species in northern and southern sub-high mountain areas in China, plays an important role in carbon fixation, water retention and material utilization, has direct effects on maintaining ecological balance, and is widely applied to protection forest and cultivation returning forest construction, and is one of main choices of artificial forest tree species. At present, the larch is mainly subjected to genetic improvement in the traditional breeding mode such as fine variety breeding, interspecific hybridization and the like in China, but the genetic improvement process is restricted to a certain extent due to long breeding period, complex asexual propagation technology and low efficiency and time and labor consumption of the traditional breeding mode, and the requirement of modern forest genetic breeding cannot be met. The invention aims to establish a set of efficient genome editing tool in larch bodies and promote the research progress of directional improvement of genetic traits of larch. With the gradual maturation of modern genetic engineering technology and the development of CRISPR-Cas genome editing technology, opportunities are brought for the rapid improvement of important characteristics of larch.

However, because of the different genomic structures and expression characteristics of different plant species, the different genome editing tools work differently in different plant species. Different promoters, cas proteins and different guide RNAs are required to be selected in different plant species to construct a gene editing expression control system that is tailored to the species. Since larch belongs to gymnosperm, genetic engineering in gymnosperm progresses slowly, no report of successful genome editing in larch body exists at present, and research and development of directional improvement of genetic characters of larch by using the emerging technology are slow. Therefore, the establishment of a set of tools capable of carrying out efficient genome editing of larch has important significance for genome engineering research and development of larch and related plants.

Disclosure of Invention

The technical problem to be solved by the invention is that a high-efficiency gene editing system which is not applicable to plants, particularly gymnosperms, particularly pine plants at present.

The technical scheme for solving the technical problem is to provide a genome editing expression frame. The genome editing expression box comprises the following elements: the promoter LarPE004, the encoding gene of the Cas9 protein, the sgRNA cloning and transcription unit are used for driving the expression of the encoding gene of the Cas9 protein and the sgRNA cloning and transcription unit by the promoter LarPE 004.

Wherein the promoter LarPE004 in the genome editing expression cassette described above is a) or b) or c) as follows:

a) The nucleotide sequence is represented by Seq ID No. 1;

b) Has more than 99%, more than 95% or more than 90% homology with the nucleotide sequence defined in a), and has promoter function

c) Or a DNA fragment which hybridizes under stringent conditions with the promoter defined in a) or b) and has a promoter function.

Wherein the Cas9 protein described in the genome editing expression cassette described above is Cas9 (lar_spry). The amino acid sequence of Cas9 (Lar_sprY) is shown as Seq ID No. 2.

Wherein, the encoding gene of the NLS protein is also connected after the encoding gene of the Cas9 protein in the genome editing expression frame.

Further, the structure of the genome editing expression frame is as follows: larPE004-Cas9-NLS-polyA-tRNA-CCDB-sgRNA scaffold-tRNA-HspT.

Wherein the Cas9 is a Cas9 encoding gene and the NLS is a nuclear localization signal; the polyA is a polyadenylation tail; the tRNA is a transfer RNA; the CCDB is a coding gene of a toxin protein CcdB; the sgRNA scaffold is a sgRNA cloning and transcription unit; the HspT is a rice HSP terminator, and the number of sgRNA cloning and transcription units is 1, 2, 3, 4, 5 or 6.

Preferably, the Cas9 protein therein is Cas9 (lar_spry). The amino acid sequence of Cas9 (Lar_sprY) is shown as Seq ID No. 2.

In the specific construction of the expression cassette or backbone vector, care should be taken that every 1 additional sgRNA cloning and transcription unit, a tRNA element is required to be separated from the adjacent sgRNA cloning and transcription unit and the last HspT region of the expression cassette.

Wherein, the nucleotide sequence of the genome editing expression frame is shown as 9bp-7427bp in the Seq ID No. 3.

The invention also provides application of the genome editing expression frame in preparing genome editing vectors.

The invention also provides a genome editing vector containing the genome editing expression frame.

Further, the above genome editing vector may be constructed based on a gene editing backbone vector in the art. For example, at least one of pTX171, pGEL031 or pZHY988 may be used.

The invention also provides the use of the genome editing expression frame or genome editing vector in performing genome editing on plants. Further, the plant is a gymnosperm or an angiosperm. Preferably, the gymnosperm is a pinaceae plant. More preferably, the pinaceae plant is larch.

The invention also provides a method for genome editing of plants. The method comprises the following steps:

a) Designing a gene editing site and sgRNA aiming at a target gene to be edited;

b) Constructing the genome editing vector of any of claims 9, wherein the sgRNA of the gene of interest of step a) is over-constructed;

c) And b) transforming plant cells or tissues by using the genome editing vector obtained in the step b), and inducing regeneration to obtain a plant with the edited genes.

Further, the plant is a gymnosperm or an angiosperm. Preferably, the gymnosperm is a pinaceae plant. More preferably, the pinaceae plant is larch.

The invention has the beneficial effects of providing an efficient expression frame for a CRISPR-Cas9 gene editing system and a corresponding gene editing expression vector. The expression frame and the corresponding vector are particularly suitable for gene editing in larch. Experiments show that the gene editing efficiency of the expression frame and the corresponding vector in larch protoplast can reach 72.5%. Meanwhile, the gene has higher gene editing activity at a plurality of PAM sites such as PAM NGG, NAG, NGT and the like, and has wide application prospect in the research direction of directional improvement of genetic traits of larch.

Drawings

FIG. 1, schematic diagram of different gene directed editing systems based on larch protoplast transient transformation system and editing activity results.

A. The vector schematic diagram of the larch gene directional editing system tested in the invention. 35S: STU Cas9_V01 System: the 35S promoter initiates Cas9 protein and guide RNA single transcription unit system; zmUBQ1: STU Cas9_V01 System: the zmebq 1 promoter initiates Cas9 protein and guide RNA single transcription unit system; larPE004 STU Cas9_V01 System: the LarPE004 promoter starts the Cas9 protein and the guide RNA single transcription unit system; larPE004 TTU Cas9_V01 System: the LarPE004 promoter initiates Cas9 protein and OsU promoter transcription of the guide RNA by the dual transcription unit system; larPE004: STU Cas9_V02 System: the LarPE004 promoter starts a Cas9 protein variant Lar_sprY and a guide RNA single transcription unit system;

35S Promoter: the cauliflower virus 35S promoter; zmebq 1 Promoter: the maize Ubiquitin promoter; larPE004 Promoter: larch promoter LarPE004; osU6 Promoter: a rice U6 promoter; cas9 (WT): a Cas9 protein gene derived from streptococcus pyogenes; cas9 (lar_spry): variants of Cas9 (WT); NLS: a nuclear localization signal); hsp-T: a terminator;

the tRNA is a transfer RNA encoding gene; the illustrated sgrnas include the sgrnas replacing ccdB elements, and the sgRNA cloning and transcription units.

B. And shearing activity high-throughput sequencing results of different gene directional editing systems based on larch protoplast transient transformation systems. 35S, STU Cas9_V01, zmUBQ1, STU Cas9_V01, larPE004, TTU Cas9_V01, and the editing activity of the four gene editing systems in LarbHLH03, larMYB06 and LarCKX01 larch endogenous genes.

C. And (3) shearing activity high-throughput sequencing results of a modified larch directional editing system based on a larch protoplast transient transformation system. ZmUBQ1:: STU Cas9_V01, larPE004:: STU Cas9_V02 edit activity at site of larch endogenous gene LarACT01-GGG-01, larACT01-GAG-01, larNAC 02-TGT-01.

FIG. 2 shows the results of the cleavage detection of the STU Cas9_V02 directional editing system on the target sites of LarACT01-GGG-01, larACT01-GAG-01 and LarNAC02-TGT-01 by using the LarPE004 based on the transient transformation system of larch protoplast.

A: editing efficiency of LarACT01-GGG-01 site cleavage detection result, control: amplifying the fragment of the wild plant; control +: the amplified fragment of the wild plant is digested with MfeI. B: editing efficiency of LarACT01-GAG-01 site cleavage detection result, control: amplifying the fragment of the wild plant; control +: the amplified fragment of the wild plant is digested with MfeI. C: editing efficiency cleavage detection result of LarNAC02-TGT-01 locus, control: amplifying the fragment of the wild plant; control +: the amplified fragment of the wild plant was digested with AclI.

FIG. 3, larPE004 based on larch protoplast transient transformation system: STU Cas9_V02 directed editing system Sanger sequencing results at LarACT01-GGG-01, larACT01-GAG-01, larNAC02-TGT-01 target sites. A: sanger sequencing results of the LarACT01-GGG-01 site. B: sanger sequencing results of the LarACT01-GAG-01 site. C: sanger sequencing results of LarNAC02-TGT-01 site.

Detailed Description

As described in the background art, as the working efficiency of different genome editing tools in different plant species is different, and different promoters are needed for the genome editing tools of different plant species to start Cas proteins and corresponding sgRNAs for genome editing, no tools for performing genome editing of larch are reported at present. The present invention is based on the creation of a viable applicable genome editing system based on CRISPR-Cas9 technology for gymnosperms, in particular for pinaceae, in particular for the plant, fall She Songda.

The invention tests the promoters from different sources to start the Cas9 protein and the guide RNA, and combines with a Single Transcription Unit (STU) CRISPR-Cas9 genome editing system to form a tool capable of realizing genome editing in larch, thereby providing a feasible genome editing scheme for larch. It was found that the best results were obtained using the novel promoter LarPE004 derived from larch to simultaneously initiate expression of Cas9-NLS protein and guide RNA. In order to establish a larch genome editing system, the invention mainly utilizes a larch protoplast transient expression technology to rapidly detect the editing efficiency of the genome editing system on the larch endogenous genes.

Meanwhile, in order to realize efficient larch genome editing activity, the invention also creatively considers that specific Cas9 variant protein Cas9 (Lar_sprY) is screened and used from the optimization and point prominence of Cas9 protein. Cas9 (lar_spry) has 11 amino acid substitutions compared to Cas9 wild type (Walton, R.T, et al science 2020).

In a preferred embodiment of the invention, an editing system LarPE004 is established, wherein the promoter LarPE004 and Cas9 variant protein Cas9 (Lar_sprY) are matched, the STU Cas9_V02 is an editing system, and the editing efficiency of editing activity in larch protoplast is up to 72.5 percent and is far higher than that of other larch genome editing systems established by contrast. Meanwhile, the LarPE004 breaks through the limit of PAM by STU Cas9_V02, and has higher gene editing activity at the position of NGG, NAG, NGT of PAM, so that the unexpected technical effect gives better application prospect to the genome editing expression frame.

The vectors in this example were specifically constructed in the following manner:

a. constructing a skeleton carrier: taking pGEL031 vector as basic skeleton, substituting LarPE004 for corn Ubi promoter, substituting Cas9 (Lar_sprY) -NLS protein coding gene for Cas9 protein coding gene, constructing CRISPR-Cas9 skeleton vector LarPE004-STU Cas9_V02;

b. constructing a directional gene editing vector: designing a gene editing site and sgRNA for a target gene to be edited, synthesizing a primer pair of the sgRNA, annealing the primer pair to form double-stranded DNA with an adhesive tail end, and replacing the ccdB element in the pGEL031 vector skeleton with the double-stranded DNA to obtain a directional editing expression vector;

and c, using the directional editing expression vector obtained in the step b to transform larch protoplast, and detecting the gene directional editing condition of the protoplast. Furthermore, when the ccdB element in pGEL031 vector backbone is replaced by using the primer pair double-stranded DNA of sgRNA in step b, the Golden Gate method cloning can be used for ligation.

The invention is further illustrated by the following specific examples.

Example 1 construction and comparison of backbone vector of larch Gene targeting modification System

1. 35S:: STU Cas9_V01, zmUBQ1:: STU Cas9_V01, larPE 004::: STU Cas9_V01 skeleton vector construction

The invention uses the reported pGEL031 (Tang, single transcript unit CRISPR 2.0systems for robust Cas9 and Cas12a mediated plant genome editing.2019,Plant Biotechnol J) vector as an initial framework vector, the vector starts the co-transcription of Cas9 protein and sgRNA unit by the maize strong promoter Ubiquitin, and the framework vector is the framework vector with the number ZmUBQ 1:STU Cas9_V01 in the example.

Using pGEL031 as a backbone, larPE004 gene fragment was amplified from larch genomic DNA using LarPE004-F and LarPE004-R (Table 1), cauliflower virus promoter 35S was amplified from pTX171 (Tang, A Single Transcript CRISPR-Cas9 System for Efficient Genome Editing in plants.2016, molecular Plant) using 35S-F and 35S-R, and the amplified LarPE004 gene fragment and 35S promoter fragment were digested with FD-AscI and FD-SbfI restriction enzymes as follows: 10X Fastdigest Green Buffer,3uL; FD-AscI,1uL; FD-SbfI,1uL; amplifying the gene fragment (2 ug), 1uL; ddH2O,24uL;37 ℃ for 1-2 h. The digested product was directly recovered using the AxyPrep DNA gel recovery kit.

Table 1 primer for constructing framework vector of gene orientation modification system and application thereof

The pGEL031 vector is digested with FD-AscI and FD-SbfI restriction enzymes as follows: 10X Fastdigest Green Buffer,3uL; FD-AscI,1uL; FD-SbfI,1uL; pGEL031 plasmid DNA (3 ug), 1uL; ddH ₂ O,24uL;37 ℃ for 1-2 h. pGEL031 enzyme fragment size is approximately2008bp and 13755bp, and recovering the fragment of about 13755bp using AxyPrep DNA gel recovery kit.

The recovered products after the gene fragments of the LarPE004 and 35S promoters are respectively digested and connected with pGEL031 vector by using T4ligase, and the connection system is 10 xT 4 library Buffer,2uL; t4ligase, 1uL; pGEL031 fragment, 2uL (50 ng); larPE004 or 35S restriction enzyme fragment, 6uL (molar quantity is 10 times of carrier); ddH ₂ O,9uL;22 ℃ for 2h. After the reaction, 6uL of the ligation product was used to transform competent cells of E.coli strain DB3.1, and LB solid plates containing Kan (50 mg/L) antibiotics were applied and cultured at 37℃for 18-22h. And (3) carrying out enzyme digestion and sequencing on the monoclonal shaking bacteria to verify the correctness of the monoclonal shaking bacteria, thereby constructing and obtaining LarPE004:: STU Cas9_V01 and 35S::: STU Cas9_V01.

2. Construction of LarPE004 TTU Cas9_V01 double-unit transcription framework vector

pZHY988 is a basic skeleton, and the vector is composed of a corn strong promoter, namely a Ubiquitin promoter, for starting Cas9 protein and a rice U6 promoter guide RNA unit. LarPE004 gene fragment of larch endogenous promoter is amplified from larch genome DNA by LarPE004-F and LarPE004-R (table 1), and the amplified LarPE004 gene fragment and pZHY988 are subjected to enzyme digestion by using FD-AscI and FD-SbfI restriction enzymes, wherein the enzyme digestion system is as follows: 10X Fastdigest Green Buffer,3uL; FD-AscI,1uL; FD-SbfI,1uL; amplified gene fragment or pZHY988 (2 ug), 1uL; ddH2O,24uL;37 ℃ for 1-2 h. The amplified LarPE004 fragment enzyme digestion products are directly recovered by an AxyPrep DNA gel recovery kit. The pZHY988 enzyme fragment sizes were about 2010bp and 13844bp, and the fragment of about 13844bp was recovered using the AxyPrep DNA gel recovery kit.

The amplified fragment enzyme digestion product and the pZHY988 vector enzyme digestion product are connected by T4ligase, and the connection system is 10 xT 4ligase Buffer,2uL; t4ligase, 1uL; pZHY988, 2uL (50 ng); larPE004 gene fragment enzyme cutting product, 6uL (molar quantity is 10 times of vector); ddH ₂ O,9uL;22 ℃ for 2h. After the reaction, 6uL of the ligation product was used to transform competent cells of E.coli strain DB3.1, and LB solid plates containing Kan (50 mg/L) antibiotics were applied and cultured at 37℃for 18-22h. Shaking the monoclonal bacteriaAnd performing enzyme digestion and sequencing to verify the correctness of the vector, thereby constructing the LarPE 004:TTU Cas9_V01 double-unit transcription skeleton vector.

3. Construction of LarPE 004:STU Cas9_V02 skeleton vector

Taking pGEL031 as a framework, larPE004 proter-Cas 9 (Lar_spRY) -NLS-polyA-tRNA-CCDB-sgRNAscaffold-tRNA-HspT gene fragment is shown as Seq ID No.3 (9 bp-2010bp is LarPE004 promoter, 2035bp-6246bp is Cas9 (Lar_sprY) -NLS,6253bp-6302bp is polyA,6303bp-6379bp, 7095bp-7171bp is tRNA,6387bp-7011bp is CCDB region, 7019bp-7094bp is sgRNA cloning and transcription unit, 7178bp-7427bp is Hsp-T terminator, both ends of Seq ID No.3 are AscI and SacI cleavage site, 1 bp-8 bp is AscI,7428-7433 is SacI), 1bp-7234bp of sequence of Seq ID No.3 is synthesized, and the synthesized fragment and pGFD-7411 bp are subjected to restriction endonuclease restriction enzyme system as follows. 10X Fastdigest Green Buffer,3uL; FD-AscI,1uL; FD-AflII,1uL; synthetic fragment or pGEL031 (2 ug), 1uL; ddH2O,24uL;37 ℃ for 1-2 h. The digested product of the synthesized fragment was directly recovered with the AxyPrep DNA gel recovery kit. pGEL031 enzyme fragment sizes were about 7149bp and 8614bp, and the fragment of about 8614bp was recovered using the AxyPrep DNA gel recovery kit.

Connecting the synthetic fragment enzyme digestion product with pGEL031 vector enzyme digestion product by using T4ligase, wherein the connecting system is 10 xT 4ligase Buffer,2uL; t4ligase, 1uL; pGEL031 fragment, 2uL (50 ng); synthesizing a fragment enzyme digestion product, and 6uL (the molar quantity is 10 times of that of the carrier); ddH ₂ O,9uL;22 ℃ for 2h. After the reaction, 6uL of the ligation product was used to transform competent cells of E.coli strain DB3.1, and LB solid plates containing Kan (50 mg/L) antibiotics were applied and cultured at 37℃for 18-22h. And (3) carrying out enzyme digestion and sequencing on the monoclonal shaking bacteria to verify the correctness of the monoclonal shaking bacteria, thereby constructing and obtaining the LarPE 004:STU Cas9_V02 skeleton vector.

Example 2 construction of endogenous Gene targeting site directed mutagenesis vector of different Larix GmbH Gene targeting editing System

Target sites are designed for LarbHLH03, larMYB06 and LarCKX01 and LarActin, larNAC02 which are endogenous genes of larch, and upstream and downstream primers are synthesized artificially (see Table 2). Mixing 20uL of each of the upstream primer and the downstream primer, denaturing at 95 ℃ for 10min, and naturally cooling and annealing to form target site double-stranded DNA with sticky ends. The annealed fragments and BsaI digested different larch genes were cloned and linked by Golden Gate method using the system backbone vector product for directed editing. The Golden Gate reaction system is as follows: 10×T4 library buffer,2uL; bsaI,1uL; t4 DNA ligase,1uL; larch gene orientation modification system framework vector enzyme cutting product (100 ng), 1uL; annealing products (10 mM), 2uL of target sites of different endogenous genes; ddH2O,13uL. The reaction procedure was as follows: (37 ℃,5min;16 ℃,10 min). Times.10 cycles; 37 ℃ for 10min;80 ℃ for 10min. After the end of the procedure, 6uL Golden Gate reaction product was added to E.coli DH5a competent cells for transformation, and the cells were plated with LB solid medium containing Kan (50 mg/L) for 18-22h. And (3) identifying positive colonies by monoclonal colony PCR (the system is as described above), and carrying out verification such as plasmid extraction, enzyme digestion, sequencing and the like on the positive colonies to obtain the directional editing expression vectors of different endogenous gene target sites.

TABLE 2 primers for constructing target site directed mutation vectors of LarHLH03, larMYB06, larCKX01 and LarActin, larNAC02 and application thereof

According to the method, expression vectors of 35S, STU Cas9_V01, zmUBQ1, STU Cas9_V01, larPE004, TTU Cas9_V01 framework vectors targeting LarHLH03, larMYB06 and LarCKX01 sites are constructed, and expression vectors of ZmUBQ1, STU Cas9_V01, larPE004, STU Cas9_V01 and LarPE004, STU Cas9_V02 targeting LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 sites are also obtained.

Example 3 detection of directed mutant Activity of different larch Gene targeting editing systems against target sites

1. Sequencing method for detecting editing activity of different larch gene directional editing systems aiming at endogenous gene loci

Through larch protoplast preparation and transient transformation system, the endogenous gene targeting site directed mutation vectors of different larch gene directed editing systems are introduced into larch protoplast cells, after culturing for 48 hours, protoplast genome DNA is extracted, high-throughput sequencing primers are designed to amplify different target gene sites respectively (as shown in Table 3), and the amplification system is as follows: 10×Taq Buffer,2.5uL; taq DNA Polymerase (2.5U/. Mu.L), 0.4uL; dNTPs (10 mM), 0.5uL; upstream detection primer F,0.5uL; downstream detection primer R,0.5uL; gDNA (100 ng/. Mu.L), 2uL; ddH2O,18.6uL. The amplification procedure was as follows: 95 ℃ for 3min; (95 ℃,30s;56 ℃,20s;72 ℃,40 s). Times.34 cycles; 72 ℃ for 5min;12℃for 5min. The PCR products were then subjected to high throughput sequencing of amplicon nova-PE150, with each amplicon sequencing read numbers above 40000reads.

TABLE 3 primers and primer uses for detecting target site directed mutations of LarHLH03, larMYB06, larCKX01, larActin, larNAC02

As shown in FIG. 1B, in the high-throughput sequencing result, compared with control, 35S is shown in the specification, STU Cas9_V01 and ZmUBQ1 are shown in the specification, STU Cas9_V01 and LarPE004 are shown in the specification, four gene orientation modification systems are shown in the specification, namely, STU Cas9_V01 and LarPE004, have the activity of orientation gene editing on LarbHLH03, larMYB06 and LarCKX01 of larch endogenous genes, and the editing activity of LarPE004 is higher than that of the STU Cas9_V01 system. The result shows that the LarPE004 STU Cas9_V01 gene directional modification system can realize the genome editing of directional knockout aiming at the endogenous locus of larch genome. Meanwhile, it is also demonstrated that the single transcription unit system is more suitable for larch gene editing than the dual-unit system, and that larch endogenous promoter LarPE004 initiates Cas9 and sgRNA activity in larch over promoters of other species.

As shown in FIG. 1C, in the high throughput sequencing result, the editing activity of modified LarPE 004:STU Cas9_V02 in larch endogenous sites LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 is far higher than that of ZmUBQ 1:STU Cas9_V01 and LarPE 004:STU Cas9_V01 systems, and the editing activity in larch protoplast reaches about 11.8% -72.5%. The result shows that the LarPE004 STU Cas9_V02 gene directional modification system can realize high-efficiency directional knockout genome editing aiming at the endogenous locus of larch genome.

2. PCR-RELP detection method for STU Cas9_V02 gene directional editing system editing activity aiming at larch endogenous gene locus

The preparation of larch protoplast and a transient transformation system are adopted, namely, the directional mutation carrier of endogenous genes of STU Cas9_V02 gene targeted LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 sites of a directional editing system is transferred into larch protoplast cells through PGE, the protoplast genome DNA is extracted after culturing, PCR amplification primers are designed to amplify different target gene sites respectively (as shown in Table 3), and the amplification system is as follows: 10×Taq Buffer,2.5uL; taq DNA Polymerase (2.5U/. Mu.L), 0.4uL; dNTPs (10 mM), 0.5uL; upstream detection primer F,0.5uL; downstream detection primer R,0.5uL; gDNA (100 ng/. Mu.L), 2uL; ddH2O,18.6uL. The amplification procedure was as follows: 95 ℃ for 3min; (95 ℃,30s;56 ℃,20s;72 ℃,40 s). Times.34 cycles; 72 ℃ for 5min;12℃for 5min. The PCR amplified products obtained were digested with MfeI, mfeI, aclI restriction enzymes, respectively, to give smaller two bands in the case of wild type and one resistant band of the same size as control (-) in the case of mutant. The cleavage activity of the LarPE 004:STU Cas9_V02 editing system was initially evaluated by detecting the amounts of the resistant bands and the cleavage bands.

As shown in FIG. 2, the activity of the STU Cas9_V02 editing system in larch endogenous loci LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 can reach 90.6%. In addition, PAM is not limited to NGG, and the activity of the sites of NAG and NGT of PAM can reach 8.6% -20.5%. The result shows that the LarPE004 STU Cas9_V02 gene directional modification system can realize genome editing of high-efficiency directional knockout aiming at larch genome, which is not limited to NGG PAM locus.

3. LarPE004 STU Cas9_V02 gene directional editing system Sanger sequencing detection method aiming at editing activity of endogenous gene locus of larch

Based on the PCR-RELP detection method, in order to determine the editing starting position of the STU Cas9_V02 editing system, the inventor cuts and recovers the resistance bands, and uses primers LarACT01-GGG-01-F2, larACT01-GAG-01-F2 and LarNAC02-TGT-01-F2 (Table 3) to carry out Sanger sequencing on the enzyme-cut resistance bands of LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 sites respectively.

Sequencing is shown in FIG. 3, where multimodal is present, namely LarPE 004:STU Cas9_V02 directional editing system begins to generate base editing. The experimental results show that: STU Cas9_V02 gene directional editing system starts editing at 3-4 positions in front of PAM position of LarACT-GGG-01 locus; editing was initiated 3-4 before the PAM position of the larct-GAG-01 site and 4-8 before the PAM position of the LarNAC02-TGT-01 site. The result shows that the initial editing position of the STU Cas9_V02 gene directional editing system is 3-8 bits, and the initial editing position of the STU Cas9 directional editing system moves away from the PAM direction.

It can be seen from a summary of the results of the above examples: the activity detection result in protoplast shows that the STU Cas9_V02 gene directional editing system shows high-efficiency shearing activity in LarACT-GGG-01, larACT-GAG-01 and LarNAC02-TGT-01 sites, namely, the editing activity of the corresponding sites is greatly improved, and the editing range of Cas9 (WT) is also widened. The invention successfully realizes efficient genome editing based on CRISPR-Cas9 in larch, proves the feasibility of larch genome editing, expands the available method for directionally improving the genetic characters of larch, and is hopeful to promote the research of related plant genome engineering.

Sequence listing

<110> university of electronic science and technology

<120> genome editing expression frame, vector and application thereof

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2002

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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ttcctttttt ggactatttt atcataaaag ttaatttcat tctagtcttc ctcttctttt 60

atacaatttt attaattgtc taactaaatt ctttaacaac aatatatttc atccatcgac 120

tttctcttat ctatcttaca atcttatatg ggtcacgaat gatagatgga gtgtccactt 180

gaaacattga atcatttcaa aacaaataaa aagtaaaatt ttagaaaatt ttattgcaaa 240

aaaattacaa aaatatacta gtaaaaaaga ttaaatttat ccatttatta actcactcca 300

ttcctcccta cctccttatg ggacctattc attttacctc cttatgggac cctctattca 360

ttttacacac cattattcat taggaagatt ggatgtatgc acattgaatc ccaaccatgg 420

tggtaaggca ggagggcaag tgtttataaa gaaaagacca ttcattttta ccatacctct 480

tatggactct attcatttta ccattattca ttagaagatt ggatgtatgc atgaatccca 540

tggtggaagg caggagcaag tgtttataaa aaagaaaatt tattaaatcg tggttatctt 600

gaatgactaa ggaaaaactc aaacattaaa ataatgaaag tactaccaca cccattcttg 660

atgccaatgc aagttagttc ctttctactt ggcatgccaa tacttacttg agaaaaaaag 720

taataaagtg atagaatgat caataatttt ggaacactaa aggctcccat aatttgtttt 780

tacttatagt taaaaaaaat aagaacataa cccctaaggc acataatttg ttttcctcat 840

catttaaaaa aaagagaaca taactcctaa agaatacctt atcacaactt gatagagata 900

tcaattgatg ataaagaagg tctaacctat ttatatctcc ctcaagtcct tgcctttgtg 960

ggctgctaaa aggctttgct gcaaggagct ttagaggccg gtgacttatt tattattata 1020

attattttaa atacctatta ttattattat aattatttta aaataattta tttaattatt 1080

aactatttgt ttgtgtggtt actttcaaca caatgtatct agacactttc ggtcaattat 1140

tgaccaaaat ttaagcttta agataattat ttgatgcaat tgatgtggta aatgatcatc 1200

agtttggcat ctgcccaacc actacagagc ccgcgggacc gcaaccttat cttttcttcg 1260

ttacggagcc agtcaaaatt ttagaaggag aatactttaa ggcggctcca ccgaccttat 1320

cccacttgca caagtggcct cctagaagct gacacctgtc ttaatatgaa tggacttcat 1380

tgggcggtcg tcgattggtg aatttaaata cggctccacc cctccttatt tttcattgtg 1440

atttcttgat ttggagagtt ttccacgacg gcaggcgaag caacagagag cgtctctatc 1500

gattgagttc aggtaacgtg ttcttctgtc tcgatttctt tctttgtatt tttaatgtct 1560

ctggttctca tttaactgtg gtaggccggc tcttcttgat cttgcttttc aatcccaaat 1620

cagaaggcta ttctctggat ctcgtttgaa ttccaaatca gaggttttat ttcgttattg 1680

tgatttattt tctggaatta ttgttttgat aaaaggtttt cgtttaattt catcggttat 1740

ggcttgatga ggaataccaa atcctatttg cttcttgtgt gatttgttgt tcctctattt 1800

ctgggtttta atggagttct tgccagtttg tgttttatag ggttttgaat ccgtgaatcc 1860

tcgacagctt gtagcctagg gtttttttgc atctgcgggt tattatcttt tattgttcag 1920

gttttcagac tatccttatt gtttcgacta tttcttatgg ttgtgcaggt ggtgggttat 1980

tctttattga aaacttcaat at 2002

<210> 2

<211> 1368

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Arg Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Leu Trp

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Lys Gln Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Arg Leu Gly Ala Pro Arg Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Pro Lys Gln Tyr Arg Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 3

<211> 7433

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggcgcgcctt ccttttttgg actattttat cataaaagtt aatttcattc tagtcttcct 60

cttcttttat acaattttat taattgtcta actaaattct ttaacaacaa tatatttcat 120

ccatcgactt tctcttatct atcttacaat cttatatggg tcacgaatga tagatggagt 180

gtccacttga aacattgaat catttcaaaa caaataaaaa gtaaaatttt agaaaatttt 240

attgcaaaaa aattacaaaa atatactagt aaaaaagatt aaatttatcc atttattaac 300

tcactccatt cctccctacc tccttatggg acctattcat tttacctcct tatgggaccc 360

tctattcatt ttacacacca ttattcatta ggaagattgg atgtatgcac attgaatccc 420

aaccatggtg gtaaggcagg agggcaagtg tttataaaga aaagaccatt catttttacc 480

atacctctta tggactctat tcattttacc attattcatt agaagattgg atgtatgcat 540

gaatcccatg gtggaaggca ggagcaagtg tttataaaaa agaaaattta ttaaatcgtg 600

gttatcttga atgactaagg aaaaactcaa acattaaaat aatgaaagta ctaccacacc 660

cattcttgat gccaatgcaa gttagttcct ttctacttgg catgccaata cttacttgag 720

aaaaaaagta ataaagtgat agaatgatca ataattttgg aacactaaag gctcccataa 780

tttgttttta cttatagtta aaaaaaataa gaacataacc cctaaggcac ataatttgtt 840

ttcctcatca tttaaaaaaa agagaacata actcctaaag aataccttat cacaacttga 900

tagagatatc aattgatgat aaagaaggtc taacctattt atatctccct caagtccttg 960

cctttgtggg ctgctaaaag gctttgctgc aaggagcttt agaggccggt gacttattta 1020

ttattataat tattttaaat acctattatt attattataa ttattttaaa ataatttatt 1080

taattattaa ctatttgttt gtgtggttac tttcaacaca atgtatctag acactttcgg 1140

tcaattattg accaaaattt aagctttaag ataattattt gatgcaattg atgtggtaaa 1200

tgatcatcag tttggcatct gcccaaccac tacagagccc gcgggaccgc aaccttatct 1260

tttcttcgtt acggagccag tcaaaatttt agaaggagaa tactttaagg cggctccacc 1320

gaccttatcc cacttgcaca agtggcctcc tagaagctga cacctgtctt aatatgaatg 1380

gacttcattg ggcggtcgtc gattggtgaa tttaaatacg gctccacccc tccttatttt 1440

tcattgtgat ttcttgattt ggagagtttt ccacgacggc aggcgaagca acagagagcg 1500

tctctatcga ttgagttcag gtaacgtgtt cttctgtctc gatttctttc tttgtatttt 1560

taatgtctct ggttctcatt taactgtggt aggccggctc ttcttgatct tgcttttcaa 1620

tcccaaatca gaaggctatt ctctggatct cgtttgaatt ccaaatcaga ggttttattt 1680

cgttattgtg atttattttc tggaattatt gttttgataa aaggttttcg tttaatttca 1740

tcggttatgg cttgatgagg aataccaaat cctatttgct tcttgtgtga tttgttgttc 1800

ctctatttct gggttttaat ggagttcttg ccagtttgtg ttttataggg ttttgaatcc 1860

gtgaatcctc gacagcttgt agcctagggt ttttttgcat ctgcgggtta ttatctttta 1920

ttgttcaggt tttcagacta tccttattgt ttcgactatt tcttatggtt gtgcaggtgg 1980

tgggttattc tttattgaaa acttcaatat cctgcaggta gatcgctcgt cgacatggac 2040

aagaagtact cgatcggcct cgatattggg actaactctg ttggctgggc cgtgatcacc 2100

gacgagtaca aggtgccctc aaagaagttc aaggtcctgg gcaacaccga tcggcattcc 2160

atcaagaaga atctcattgg cgctctcctg ttcgacagcg gcgagacggc tgagaggacg 2220

cggctcaagc gcaccgcccg caggcggtac acgcgcagga agaatcgcat ctgctacctg 2280

caggagattt tctccaacga gatggcgaag gttgacgatt ctttcttcca caggctggag 2340

gagtcattcc tcgtggagga ggataagaag cacgagcggc atccaatctt cggcaacatt 2400

gtcgacgagg ttgcctacca cgagaagtac cctacgatct accatctgcg gaagaagctc 2460

gtggactcca cagataaggc ggacctccgc ctgatctacc tcgctctggc ccacatgatt 2520

aagttcaggg gccatttcct gatcgagggg gatctcaacc cggacaatag cgatgttgac 2580

aagctgttca tccagctcgt gcagacgtac aaccagctct tcgaggagaa ccccattaat 2640

gcgtcaggcg tcgacgcgaa ggctatcctg tccgctaggc tctcgaagtc tcggcgcctc 2700

gagaacctga tcgcccagct gccgggcgag aagaagaacg gcctgttcgg gaatctcatt 2760

gcgctcagcc tggggctcac gcccaacttc aagtcgaatt tcgatctcgc tgaggacgcc 2820

aagctgcagc tctccaagga cacatacgac gatgacctgg ataacctcct ggcccagatc 2880

ggcgatcagt acgcggacct gttcctcgct gccaagaatc tgtcggacgc catcctcctg 2940

tctgatattc tcagggtgaa caccgagatt acgaaggctc cgctctcagc ctccatgatc 3000

aagcgctacg acgagcacca tcaggatctg accctcctga aggcgctggt caggcagcag 3060

ctccccgaga agtacaagga gatcttcttc gatcagtcga agaacggcta cgctgggtac 3120

attgacggcg gggcctctca ggaggagttc tacaagttca tcaagccgat tctggagaag 3180

atggacggca cggaggagct gctggtgaag ctcaatcgcg aggacctcct gaggaagcag 3240

cggacattcg ataacggcag catcccacac cagattcatc tcggggagct gcacgctatc 3300

ctgaggaggc aggaggactt ctaccctttc ctcaaggata accgcgagaa gatcgagaag 3360

attctgactt tcaggatccc gtactacgtc ggcccactcg ctaggggcaa ctcccgcttc 3420

gcttggatga cccgcaagtc agaggagacg atcacgccgt ggaacttcga ggaggtggtc 3480

gacaagggcg ctagcgctca gtcgttcatc gagaggatga cgaatttcga caagaacctg 3540

ccaaatgaga aggtgctccc taagcactcg ctcctgtacg agtacttcac agtctacaac 3600

gagctgacta aggtgaagta tgtgaccgag ggcatgagga agccggcttt cctgtctggg 3660

gagcagaaga aggccatcgt ggacctcctg ttcaagacca accggaaggt cacggttaag 3720

cagctcaagg aggactactt caagaagatt gagtgcttcg attcggtcga gatctctggc 3780

gttgaggacc gcttcaacgc ctccctgggg acctaccacg atctcctgaa gatcattaag 3840

gataaggact tcctggacaa cgaggagaat gaggatatcc tcgaggacat tgtgctgaca 3900

ctcactctgt tcgaggaccg ggagatgatc gaggagcgcc tgaagactta cgcccatctc 3960

ttcgatgaca aggtcatgaa gcagctcaag aggaggaggt acaccggctg ggggaggctg 4020

agcaggaagc tcatcaacgg cattcgggac aagcagtccg ggaagacgat cctcgacttc 4080

ctgaagagcg atggcttcgc gaaccgcaat ttcatgcagc tgattcacga tgacagcctc 4140

acattcaagg aggatatcca gaaggctcag gtgagcggcc agggggactc gctgcacgag 4200

catatcgcga acctcgctgg ctcgccagct atcaagaagg ggattctgca gaccgtgaag 4260

gttgtggacg agctggtgaa ggtcatgggc aggcacaagc ctgagaacat cgtcattgag 4320

atggcccggg agaatcagac cacgcagaag ggccagaaga actcacgcga gaggatgaag 4380

aggatcgagg agggcattaa ggagctgggg tcccagatcc tcaaggagca cccggtggag 4440

aacacgcagc tgcagaatga gaagctctac ctgtactacc tccagaatgg ccgcgatatg 4500

tatgtggacc aggagctgga tattaacagg ctcagcgatt acgacgtcga tcatatcgtt 4560

ccacagtcat tcctgaagga tgactccatt gacaacaagg tcctcaccag gtcggacaag 4620

aaccggggca agtctgataa tgttccttca gaggaggtcg ttaagaagat gaagaactac 4680

tggcgccagc tcctgaatgc caagctgatc acgcagcgga agttcgataa cctcacaaag 4740

gctgagaggg gcgggctctc tgagctggac aaggcgggct tcatcaagag gcagctggtc 4800

gagacacggc agatcactaa gcacgttgcg cagattctcg actcacggat gaacactaag 4860

tacgatgaga atgacaagct gatccgcgag gtgaaggtca tcaccctgaa gtcaaagctc 4920

gtctccgact tcaggaagga tttccagttc tacaaggttc gggagatcaa caattaccac 4980

catgcccatg acgcgtacct gaacgcggtg gtcggcacag ctctgatcaa gaagtaccca 5040

aagctcgaga gcgagttcgt gtacggggac tacaaggttt acgatgtgag gaagatgatc 5100

gccaagtcgg agcaggagat tggcaaggct accgccaagt acttcttcta ctctaacatt 5160

atgaatttct tcaagacaga gatcactctg gccaatggcg agatccggaa gcgccccctc 5220

atcgagacga acggcgagac gggggagatc gtgtgggaca agggcaggga tttcgcgacc 5280

gtcaggaagg ttctctccat gccacaagtg aatatcgtca agaagacaga ggtccagact 5340

ggcgggttct ctaaggagtc aattaggcct aagcggaaca gcgacaagct catcgcccgc 5400

aagaaggact gggatccgaa gaagtacggc gggttcctgt ggcccactgt ggcctactcg 5460

gtcctggttg tggcgaaggt tgagaagggc aagtccaaga agctcaagag cgtgaaggag 5520

ctgctgggga tcacgattat ggagcgctcc agcttcgaga agaacccgat cgatttcctg 5580

gaggcgaagg gctacaagga ggtgaagaag gacctgatca ttaagctccc caagtactca 5640

ctcttcgagc tggagaacgg caggaagcgg atgctggctt ccgctaagca gctgcagaag 5700

gggaacgagc tggctctgcc gtccaagtat gtgaacttcc tctacctggc ctcccactac 5760

gagaagctca agggcagccc cgaggacaac gagcagaagc agctgttcgt cgagcagcac 5820

aagcattacc tcgacgagat cattgagcag atttccgagt tctccaagcg cgtgatcctg 5880

gccgacgcga atctggataa ggtcctctcc gcgtacaaca agcaccgcga caagccaatc 5940

agggagcagg ctgagaatat cattcatctc ttcaccctga cgaggctcgg cgcccctagg 6000

gctttcaagt acttcgacac aactatcgat ccgaagcagt acaggagcac taaggaggtc 6060

ctggacgcga ccctcatcca ccagtcgatt accggcctct acgagacgcg catcgacctg 6120

tctcagctcg ggggcgacac tagttccggc ggcagcccaa agaagaagcg gaaggtgtct 6180

ggaggttctc ctaagaaaaa gagaaaagtg tccggcggct cccctaagaa gaagaggaag 6240

gtttgaggat ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 6300

aaaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt 6360

tcgattcccg gctggtgcag gagaccttat attccccaga acatcaggtt aatggcgttt 6420

ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggaaaccggc 6480

acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg 6540

taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc 6600

cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg ataacggctc 6660

tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct cgtcagcaaa 6720

agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat tttccgcttt 6780

ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta ccagaccgga 6840

gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac tgtcactgta 6900

atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata tcagtatata 6960

ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct tggtctcagt 7020

tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 7080

caccgagtcg gtgcaacaaa gcaccagtgg tctagtggta gaatagtacc ctgccacggt 7140

acagacccgg gttcgattcc cggctggtgc aggatccata tgaagatgaa gatgaaatat 7200

ttggtgtgtc aaataaaaag cttgtgtgct taagtttgtg tttttttctt ggcttgttgt 7260

gttatgaatt tgtggctttt tctaatatta aatgaatgta agatcacatt ataatgaata 7320

aacaaatgtt tctataatcc attgtgaatg ttttgttgga tctcttctgc agcatataac 7380

tactgtatgt gctatggtat ggactatgga atatgattaa agataaggag ctc 7433

<210> 4

<211> 58

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gagtgtcgtg ctccaccatg ggcgcgcctt ccttttttgg actattttat cataaaag 58

<210> 5

<211> 54

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gtcgacgagc gatctacctg caggatattg aagttttcaa taaagaataa ccca 54

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tgcattgtcc atcttcgaca catt 24

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aaacaatgtg tcgaagatgg acaa 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gtgtttgtcc atcttcgaca catt 24

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tgcagctttt tctgtgtgtt gtat 24

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

aaacatacaa cacacagaaa aagc 24

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gtgtgctttt tctgtgtgtt gtat 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tgcaattata aaacggaatc tgat 24

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aaacatcaga ttccgtttta taat 24

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gtgtattata aaacggaatc tgat 24

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tgcaacttcc tgtgaacaat tga 23

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cccatcaatt gttcacagga agt 23

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tgcaagcact tcctgtgaac aatt 24

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cccaaattgt tcacaggaag tgct 24

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tgcacggtat gaggaaaacg ttgg 24

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

aaacccaacg ttttcctcat accg 24

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

tgggagaaag cctgcaaatg g 21

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ctttacactg tcaatttgag tctgc 25

<210> 23

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

caccttctgg ctttttctgt ttg 23

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

cgatgaaaca atgagtaact a 21

<210> 25

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ggtttagtgc accgtatcca actg 24

<210> 26

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

cagtggacct ggaaaaaatc ctct 24

<210> 27

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

gagcattgta tgttgtttat tggttc 26

<210> 28

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

ttatatggag gactagcatt gtaaag 26

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

ggttcttaat ttgatcacgg aaa 23

<210> 30

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

atcctctctt tacaatgcta gtcc 24

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

ggataaaaag tatccaacgg gcac 24

<210> 32

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

tgaggaggag cgttctcgtt cgtt 24

<210> 33

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

ggcgcgccag atttgccttt tcaatttcag aaag 34

<210> 34

<211> 44

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

cgatctacct gcaggagtcc cccgtgttct ctccaaatga aatg 44

Claims (11)

1. A genome editing expression cassette comprising the following elements: the promoter LarPE004, the Cas9 protein coding gene and the sgRNA scaffold are driven by the promoter LarPE004; the nucleotide sequence of the promoter LarPE004 is shown as Seq ID No. 1.

2. The genome editing expression cassette of claim 1, wherein: the amino acid sequence of the Cas9 protein is shown as Seq ID No. 2.

3. The genome editing expression cassette of claim 1, wherein: and the encoding gene of the NLS protein is also connected behind the encoding gene of the Cas9 protein.

4. The genome editing expression cassette according to claim 1, characterized in that it has the structure: the promoter LarPE004-Cas9-NLS-polyA-tRNA-CCDB-sgRNA scaffold-tRNA-HspT;

the Cas9 is a Cas9 encoding gene, and the NLS is a nuclear localization signal; the polyA is a polyadenylation tail; the tRNA is a transfer RNA; the CCDB is a coding gene of a toxin protein CcdB; the HspT is a rice HSP terminator; the sgRNA scaffold is 1, 2, 3, 4, 5 or 6.

5. Use of the genome editing expression cassette of any of claims 1 to 4 in the preparation of a genome editing vector.

6. A genome editing vector comprising the genome editing expression cassette of any one of claims 1 to 4.

7. Use of the genome editing expression cassette of any one of claims 1 to 4 or the genome editing vector of claim 6 for performing plant gene editing.

8. The use according to claim 7, characterized in that the plant is a gymnosperm or an angiosperm.

9. The use according to claim 8, characterized in that the gymnosperm is a pinaceae plant.

10. Use according to claim 9, characterized in that the pinaceae plant is larch.

11. A method of genome editing of a plant, comprising the steps of:

a) Designing a gene editing site and sgRNA aiming at a target gene to be edited;

b) Constructing the genome editing vector of claim 6, wherein the sgRNA into which the gene of interest of step a) is constructed;

c) And b) transforming plant cells or tissues by using the genome editing vector obtained in the step b), and inducing regeneration to obtain a plant with the edited genes.