Method for site-specific mutagenesis of Medicago sativa gene by using CRISPR / Cas9 system

Abstract

A method for site-specific mutagenesis of a medicago sativa gene by using a CRISPR / Cas9 system. The method comprises: first constructing a universal binary expression vector MsCRISPR / Cas9 mediated and transformed by agrobacterium tumefaciens in medicago sativa; then designing a CRISPR / Cas9-based sgRNA sequence for a target gene in the medicago sativa, and connecting a DNA fragment for encoding the sgRNA sequence to MsCRISPR / Cas9 to construct a carrier MsCRISPR / Cas9::target; and then mediating and transforming the medicago sativa by using the agrobacterium tumefaciens, and obtaining, by screening, a mutant transformed plant with the target gene knocked out. According to the method, an MtU6 promoter is used for initiating sgRNA transcription in the medicago sativa.

Description

A method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system Technical Field

[0001] This invention belongs to the field of gene editing technology, and in particular relates to a method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system. Background Technology

[0002] Alfalfa (Medicago sativa.) is a widely cultivated legume forage crop, rich in high-quality dietary fiber, edible protein, various vitamins (including vitamins B, C, and E), beneficial minerals, and bioactive components such as saponins, flavonoids, carotenoids, and phenolic acids. Its high protein content and palatability make it significant for improving livestock feed quality. Besides its role as a forage crop, alfalfa's extensive root system plays a crucial role in preventing soil erosion and environmental management. Furthermore, due to the presence of numerous nitrogen-fixing microorganisms in its roots, alfalfa is also significant in improving soil fertility. Research on alfalfa gene function helps improve its traits and aids in the breeding of new alfalfa varieties with superior characteristics. Using gene silencing technology to suppress the expression of target genes in alfalfa is a highly effective method for studying gene function and breeding new varieties.

[0003] Although spontaneous mutations frequently occur in nature, the mutation frequency is extremely low; the mutation rate of most gene loci in each generation is approximately 10. -5 Or 10 -6Only mutations occurring in gametes can be passed on to the next generation. Mutagenesis methods such as chemical mutagenesis, physical mutagenesis, and DNA insertion mutagenesis can quickly produce valuable mutants from plant materials. Mutagenic factors induce mutations with a high frequency, hundreds or even thousands of times higher than spontaneous mutations, and the range and types of induced mutations are broad and diverse, sometimes even producing novel mutations rare or never before seen in nature. However, mutations introduced through mutagenesis are random, making it difficult to obtain homozygous mutants at a specific site. Similarly, only mutations occurring in gametes can be passed on to the next generation. Targeted gene silencing technology is an effective means of studying the function of specific genes. Currently, RNAi (RNA interference), as well as gene editing technologies such as ZFN, TALLEN, and CRISPR / Cas9, have been developed for targeted gene silencing. RNAi technology mainly degrades the mRNA sequence transcribed from the target gene, downregulating the translation level of mRNA, thereby inhibiting the expression of the target gene. However, when RNAi technology reduces the expression of a target gene, the gene sequence of the target gene is not mutated, and it may still be expressed to some extent. Conversely, gene silencing achieved using ZFN, TALLEN, and CRISPR / Cas9 technologies mutates the gene sequence at the DNA level, causing the gene to completely lose its function. Compared to RNAi, these three technologies are more stable and efficient. Among the three technologies, TALEN and ZFN require assembling amino acid sequence elements that recognize different bases at each target site according to the target sequence. Each target site has its own unique protein sequence, making the process time-consuming, labor-intensive, and costly. Unlike TALLEN and ZFN, CRISPR / Cas9 technology recognizes target sites through the target sequence on sgRNA. The target sequence and target site recognize each other through base complementarity pairing, which is simpler, cheaper, and easier to operate. Therefore, since CRISPR / Cas9 technology was first applied to gene editing in 2013, it has experienced rapid and widespread development. The CRISPR / Cas9 system is an acquired immune system derived from the genus Streptococcus, capable of defending against the invasion of foreign genes. It primarily consists of CRISPR and the specific Cas9 protein. CRISPR is a cluster of regularly spaced short palindromic repeats, a novel type of DNA repetitive sequence, composed of a series of short, highly conserved forward repeats interspersed with spacers of similar length. The Cas9 protein is a multidomain protein composed of 1409 amino acids, containing two nuclease domains: RuvC-like and HNH. A CRISPR / Cas9-based genome editing system comprises two parts: sgRNA and the Cas9 enzyme.This system can specifically recognize target sequences adjacent to the PAM (NGG) motif and cleave them 3 nt upstream to form a DSB (double strain break). Subsequently, the body repairs the damaged DNA through its own repair mechanisms, such as homologous recombination (HR) and non-homologous end joining (NHEJ). The NHEJ repair process can generate indels (including deletions and insertions), which in turn lead to frameshift mutations in the DNA coding sequence.

[0004] Alfalfa is a strictly cross-pollinated, autotetraploid plant with four alleles at each locus. Ideally, to obtain a stable, heritable homozygous mutant strain targeting a specific locus, multiple strains with simultaneous mutations in all four alleles would be obtained, enabling hybridization and stable inheritance among these strains. Another approach is to repeatedly hybridize naturally occurring or induced heterozygous mutants to obtain a stable, heritable homozygous mutant strain, but this is a time-consuming, labor-intensive, high-investment, and inefficient process. Therefore, obtaining stable, heritable homozygous mutants from naturally occurring or induced mutants through hybridization in alfalfa is particularly difficult, severely hindering alfalfa research and breeding, especially for recessive gene mutations where phenotypic changes are only expressed in homozygous individuals. Developing efficient targeted mutagenesis techniques in alfalfa to obtain stable, heritable homozygous mutants within a single generation is of great significance for alfalfa research and breeding. Currently, the only known method for gene editing in alfalfa is RNAi (RNAi) to reduce gene expression. There are no reports of obtaining homozygous mutants using gene editing technologies such as ZFN, TALEN, or CRISPR / Cas9. As mentioned above, compared to gene editing technologies like ZFN, TALEN, and CRISPR / Cas9, RNAi's incomplete silencing and instability limit its application. While stable, heritable homozygous mutants have been successfully obtained using CRISPR / Cas9 in species such as rice, Arabidopsis, tomato, poplar, white poplar, apple, wheat, corn, and soybean, there are no reports of gene editing using targeted gene editing technologies like CRISPR / Cas9 in alfalfa.

[0005] Summary of the Invention

[0006] To achieve gene editing in the autotetraploid plant alfalfa and apply it to production, we developed a method for site-directed mutagenesis using the CRISPR / Cas9 system. This method achieved precise site-directed gene mutations in alfalfa with a mutation rate as high as 52%, and obtained homozygous mutant plants with ideal phenotypes and silenced four alleles (homozygous mutation rate of 12%). This pioneering work establishes an efficient targeted gene editing system in alfalfa, which is of great significance for alfalfa research and breeding. Therefore, this invention aims to provide a method for site-directed gene mutagenesis in alfalfa using the CRISPR / Cas9 system.

[0007] The technical solution of the present invention is as follows:

[0008] A method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system, characterized by the following steps:

[0009] Step (1) Construct MsCRISPR / Cas9, a universal binary expression vector for alfalfa mediated by Agrobacterium tumefaciens;

[0010] Step (2) Design a CRISPR / Cas9-based sgRNA targeting sequence for the target gene in alfalfa, and connect the DNA fragment containing the sgRNA targeting sequence to the universal alfalfa vector MsCRISPR / Cas9 to construct the vector MsCRISPR / Cas9::target;

[0011] Step (3) uses Agrobacterium tumefaciens to mediate the transformation of alfalfa, thereby achieving site-directed mutation of specific genes in alfalfa.

[0012] Furthermore, the alfalfa includes wild varieties, local varieties, bred varieties, and introduced varieties.

[0013] Furthermore, the target gene includes any biologically functional gene or DNA sequence in the alfalfa genome.

[0014] Furthermore, the full sequence of the binary expression vector MsCRISPR / Cas9 for expressing the CRISPR / Cas9 system in alfalfa is shown in SEQ ID NO.1.

[0015] This technology relies on the transformation of alfalfa using Agrobacterium tumefaciens. Through transformation, T-DNA sequences containing the selection gene Hpt expression cassette, sgRNA expression cassette, and Cas9 protein expression cassette are integrated into the alfalfa genome to obtain transgenic plants. The transformed exogenous expression elements are expressed in alfalfa, thus functioning and resulting in gene knockout plants. A MsCRISPR / Cas9 expression vector was constructed to target the Agrobacterium-mediated transformation method.

[0016] Preferably, the backbone vector of the binary expression vector MsCRISPR / Cas9 for expressing the CRISPR / Cas9 system in alfalfa is the pCambia1300 vector. The pCambia1300 backbone vector has a T-DNA region for plant transformation. This T-DNA region includes a left-bound repeat (LB repeat), a right-bound repeat (RB repeat), and transformation sequences within the left and right boundary sequences. The transformation sequences within the left and right boundary sequences of the T-DNA region include an Hpt gene expression element expressing the protein against the plant selection agent hygromycin, an expression element expressing the Cas9 protein, an expression element expressing sgRNA, and a target sequence between two Aar1 restriction sites within the sgRNA expression element. The Hpt gene expression element contains a Camv 35S promoter (enhanced) promoter sequence to initiate Hpt gene transcription, a CDS sequence expressing the Hpt gene, and a Camv poly(A) termination sequence to terminate Hpt gene transcription. The Cas9 protein expression element contains a 2xCaMV 35S sequence to initiate Cas9 sequence transcription. The expression element expressing sgRNA contains a promoter sequence, a DNA sequence expressing Cas9, and a Nos terminator sequence terminating Cas9 transcription. The expression element expressing sgRNA contains an alfalfa MtU6 promoter sequence that initiates transcription of the sgRNA sequence and a DNA sequence containing the sgRNA.

[0017] In this vector (Figure 1), LB T-DNA repeat / RB T-DNA repeat represents the left and right boundary repeat sequences of T-DNA; CaMV 35s promoter / MtU6 promoter / 2x CaMV 35s promoter represents promoter elements; Hpt / sgRNA / Cas9 represents gene sequences; CaMV poly(A) / NosT represents the termination sequence; and AarI represents the sgRNA target sequence insertion site. As shown in Figure 1: sgRNA expression is initiated using the alfalfa U6 promoter; Cas9 gene expression is initiated using the 2X CaMV35S promoter; and the selection marker gene Hpt is initiated using the CaMV35S (Enhanced) promoter.

[0018] Furthermore, the sequence of the MtU6 promoter is shown in SEQ ID NO.2.

[0019] Preferably, the DNA sequence expressing sgRNA is as shown in SEQ ID NO.3, and the sequence from position 1 to position 30 of the DNA sequence expressing sgRNA contains two AarI restriction endonuclease recognition sites, which are used to construct a site-directed mutagenesis expression vector for the target gene.

[0020] Furthermore, the method for constructing the expression vector MsCRISPR / Cas9::target is as follows:

[0021] Step (1) Design a pair of oligo primers (18-24 bp) based on the target sequence to form the following DNA fragment with adapters at both ends, and synthesize it:

[0022] 5'-TTTGN 16-23 -3'

[0023] 3'-CN 16-23 CAA A-5';

[0024] Step (2) Anneal the synthesized primer pair to form a complementary DNA double-stranded fragment with sticky ends;

[0025] Step (3) The binary expression vector MsCRISPR / Cas9 was digested with AarI restriction endonuclease; after dephosphorylation by CIP reaction, it was separated by electrophoresis on a 0.8-1.2% agarose gel and the linear vector was recovered and purified.

[0026] Step (4) The annealed complementary DNA double-stranded fragment with sticky ends is ligated with the recovered and purified MsCRISPR / Cas9 linear vector in a 16°C water bath for 10-16 h using T4 DNA ligase.

[0027] Step (5) The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method. After recovery, the cells were plated on LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12-16 h.

[0028] Step (6) Pick a single colony clone and place it in 1.5 ml of LB liquid medium containing 50 mg / L kanamycin, and shake it on a shaker at 37°C for 12-16 h;

[0029] Step (7) Sequencing of the single-clone bacterial culture was performed using sequencing primer MtU6-TF. The correct target site was selected and ligated into 50-100 ml of LB liquid medium containing 50 mg / L kanamycin. The culture was shaken at 37°C for 12-16 h, and the plasmid was extracted.

[0030] Furthermore, the sequence of the sequencing primer MtU6-TF is shown in SEQ ID NO.4.

[0031] Preferably, the method for obtaining mutant plants by Agrobacterium tumefaciens-mediated transformation of alfalfa includes the following steps:

[0032] Step (1) Recovery of Agrobacterium tumefaciens strain; spread the electroporated Agrobacterium strain on YM solid plate medium and incubate at 25-29℃ for 24-48h; pick single clones and inoculate them into 30-50ml YM liquid medium and shake on a shaker at 100-220r / min at 25-29℃ for 24-48h; both YM solid medium and YM liquid medium contain 30-60mg / L kanamycin and 200-450mg / L rifampin;

[0033] Step (2) Obtaining alfalfa callus: The process of obtaining alfalfa callus is as follows:

[0034] Select plump, round alfalfa seeds, soak them in 65-80% alcohol for 1-3 minutes, wash them with sterile water 2-4 times, 0.5-2 minutes each time; soak them in 0.05-0.15% mercuric chloride and shake them by hand for 6-12 minutes, wash them with sterile water 3-5 times, inoculate them on MS solid medium, and germinate them in a light incubator or culture room for 7-16 days;

[0035] Take the germinating cotyledons and hypocotyls, cut them into small pieces, and inoculate them into callus induction medium. Incubate in the dark at 25±1℃ for 2-4 days. The callus induction medium consists of: SH basal medium + 1.0-3.0 mg / L 2,4-D (2,4-difluorophenoxyacetic acid) + 0.1-0.3 mg / L KT (kinetin) + 0.1-0.5 mg / L hydrolyzed complexin + 15-40 g / L sucrose + 5.8-9 g / L agar. Preferably, the callus induction medium consists of: SH basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 0.3 mg / L hydrolyzed complexin + 30 g / L sucrose + 8 g / L agar.

[0036] Step (3) Agrobacterium tumefaciens infection of alfalfa callus: The process of Agrobacterium tumefaciens infection of alfalfa callus is as follows: 1-2 days in advance, the Agrobacterium tumefaciens strain revived in step (1) is inoculated into 50-100 mL of YM liquid medium (Sigma), and cultured on a shaker at 25-29℃ at 100-220 r / min until the OD value of 260 / 280 is between 0.5-0.8; the bacterial solution is transferred to a 50 mL sterile centrifuge tube, centrifuged at 0-8℃ at 3000-4500 r / min for 10-15 minutes, the centrifuge tube is removed, the supernatant is discarded, and resuspended with resuspension solution until the OD value is 260 / 280. Add 50-150 μmol / L acetylsuccinone to the 0 / 280 solution between 0.5 and 0.8, and use MS liquid culture medium as the resuspension. Collect the callus induced in step (2) into a sterile Erlenmeyer flask with a breathable plastic sealing film, pour in the resuspension of Agrobacterium tumefaciens, seal the flask with the sealing film provided with the Erlenmeyer flask, and evacuate to 0.5 kPa in a vacuum pump for a total of 0.5-1.5 h, gently shaking it every 10-15 min during this period. Take out the Erlenmeyer flask and shake it on a shaker at 100-150 r / min at 25-29℃ for 0.5-1.5 h. Pour out the dried bacterial solution and spread the material on a sterile petri dish lined with filter paper to dry.

[0037] Step (4) Co-culture of alfalfa callus infected with Agrobacterium tumefaciens: The co-culture process is as follows: The dried infected material is inoculated into a co-culture medium (a sterile filter paper is placed in the medium), and cultured in the dark at 25±1℃ for 2-5 days; The composition of the co-culture medium is: MS basal medium + 1-3 mg / L 2,4-D + 0.1-0.3 mg / L KT + 15-40 g / L sucrose + 5.8-9 g / L agar + 50-150 μmol / L acetosyringone; Preferably, the composition of the co-culture medium is: MS basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 30 g / L sucrose + 8 g / L agar + 100 μmol / L acetosyringone;

[0038] Step (5) Screening and culture of alfalfa callus: The screening and culture process is as follows: the co-cultured material is inoculated into the screening medium and cultured under light in an incubator or culture room at 25±1℃ for 30-60 days; the screening medium consists of: SH basal medium + 1-3 mg / L 2,4-D + 0.1-0.3 mg / L KT + 15-40 g / L sucrose + 5.8-9 g / L agar + 150-450 mg / L cefotaxime + 150-450 mg / L carbenicillin + 10-50 mg / L hygromycin; preferably, the screening medium consists of: SH basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 250 mg / L carbenicillin + 15 mg / L hygromycin;

[0039] Step (6) Alfalfa callus differentiation culture: The screened and cultured materials are transferred to differentiation medium and cultured under light in an incubator or culture room at 25±1℃ for 15-30 days; the differentiation medium consists of: UM basal medium + 0.5-5 g / L hydrolyzed complexin + 0.1-2 mg / L kinetin + 15-40 g / L sucrose + 5.8-9 g / L agar + 150-450 mg / L cefotaxime + 4-10 mg / L hygromycin; preferably, the differentiation medium consists of: UM basal medium + 2 g / L hydrolyzed complexin + 0.4 mg / L kinetin + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 5 mg / L hygromycin;

[0040] Step (7) Rooting culture of alfalfa regenerated shoots: The differentiated 1-3cm shoots are transferred to the rooting medium, the composition of which is: MS basal medium + 0.5-2mg / L indolebutyric acid + 15-40g / L sucrose + 5.8-9g / L agar + 150-450mg / L cefotaxime; preferably, the composition of the rooting medium is: MS basal medium + 1mg / L indolebutyric acid + 30g / L sucrose + 8g / L agar + 250mg / L cefotaxime;

[0041] Step (8) Screening and detection of alfalfa mutants. The methods for screening and detecting alfalfa mutants include PCR-RE, T7E1 restriction enzyme digestion detection, and target site deep sequencing. The specific detection methods are as follows:

[0042] PCR-RE detection: DNA samples were extracted from the regenerated plants obtained above, and amplification was performed using specific primers designed for the target gene sequence. If the amplified fragment contains a specific restriction enzyme cleavage site at the Cas9 protein cleavage site, the mutated sequence cannot be cleaved by the restriction enzyme due to the loss of its cleavage site. The specific operation is as follows: a. Amplify the target gene fragment of the regenerated plant and control plant using specific primers designed for the target gene; b. Recover the amplified target fragment and digest it with a target-specific restriction enzyme; c. Detect the digestion products by agarose gel electrophoresis. Analysis of the electrophoresis results: If, after restriction enzyme digestion, the amplified product of the regenerated seedling shows a band of the same size as the undigested control sequence and no other bands, while the digested control sequence is cut into two smaller bands, it indicates that the target gene has been knocked out and the regenerated seedling is a homozygous mutant; if, after restriction enzyme digestion, the amplified product of the regenerated seedling shows a band of the same size as the undigested control sequence and no other bands, while the digested control sequence is cut into two smaller bands, it indicates that the target gene has been knocked out and the regenerated seedling is a homozygous mutant; if, after restriction enzyme digestion, the amplified product of the regenerated seedling shows a band of the same size as the undigested control sequence and no other bands, it indicates that the target gene has been knocked out and the regenerated seedling is a homozygous mutant. If the bands of the undigested control sequence are the same size, and one band is the same size as the two smaller bands of the control sequence after restriction endonuclease digestion, it indicates that the target gene has been knocked out and the regenerated seedling is a heterozygous mutation. If, after restriction endonuclease digestion, the amplified product of the regenerated seedling has no bands in the electrophoresis that are the same size as the undigested control sequence, and the digested band is the same size as the two smaller bands of the control sequence after restriction endonuclease digestion, it indicates that the target gene has not been knocked out. d. The undigested bands are recovered and ligated into pMD19-T via TA cloning. After transformation into E. coli, a batch of single clones are selected for sequencing to analyze the mutation type at the target site.

[0043] T7E1 Detection: DNA samples were extracted from the regenerated plants obtained above, and specific amplification primers were designed for the target gene sequence. If the amplified target gene fragment did not contain a restriction endonuclease site at the Cas9 protein cleavage site, the mutant plant was detected by T7E1 digestion. The specific procedure was as follows: a. Design primers to amplify the target gene sequence; b. Mix the target gene amplification products from wild-type and regenerated plants; c. Denature and renature the mixed product; d. Digest the renatured product with T7E1 enzyme (NEB) and detect it by agarose gel electrophoresis. Results analysis: If the target gene in the regenerated plant is mutated, after denaturation and renaturation, it will form a heteroduplex nucleic acid molecule with the amplification product of the wild-type plant. This heteroduplex nucleic acid molecule can be cleaved by the T7E1 enzyme to obtain two bands of ideal size; e. The PCR product of the regenerated seedling that can be cleaved by the T7E1 enzyme is TA cloned and ligated into the pMD19-T vector (Takara). After transformation into E. coli, a batch of single clones are selected for sequencing to analyze the mutation type at the target site and whether the regenerated seedling is homozygous knockout.

[0044] Target deep sequencing detection: DNA samples were extracted from the regenerated plants obtained above, and specific amplification primers were designed for the target gene sequence for amplification. The PCR product was TA cloned and ligated into the pMD19-T vector, transformed into E. coli, and a batch of single clones were selected for target deep sequencing to analyze whether there were mutations at the target sites and to analyze the mutation types.

[0045] Furthermore, this invention also protects the application of the method of site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system in alfalfa breeding and gene-edited alfalfa breeding.

[0046] This invention successfully established a CRISPR / Cas9-based gene editing technology system in alfalfa for the first time, successfully knocking out the target gene and obtaining homozygous mutant plants with distinct phenotypes. To achieve gene editing in the autotetraploid plant alfalfa, this invention, based on the existing platform of Agrobacterium tumefaciens transformation of alfalfa to obtain transgenic plants, uses the Agrobacterium transformation method to integrate the expression cassette of the Hpt gene (containing a protein expressing the selection agent hygromycin), the sgRNA expression cassette, and the T-DNA region of the Cas9 protein expression cassette into the alfalfa genome. These elements are transcribed, translated, and function within alfalfa cells, and through screening, mutant plants with the target gene knocked out are obtained. The key to introducing the CRISPR / Cas9 system into alfalfa via transgenic methods lies in finding suitable promoters to initiate the expression of each element. Among them, the CaMV 35S (Enhanced) promoter for initiating Hpt gene transcription and the 2xCaMV 35S promoter for initiating Cas9 gene transcription have been proven to initiate the transcription of related genes and perform their functions in alfalfa. The promoters for initiating sgRNA transcription are generally U6 or U3 promoters, but there are no reports on the study of U6 / U3 promoters in alfalfa. In order to efficiently initiate sgRNA transcription in alfalfa, through literature review, the MtU6 promoter, which has been studied more extensively in alfalfa and is a closely related species of alfalfa, was selected to initiate sgRNA transcription.

[0047] Compared with existing techniques for downregulating gene expression using RNAi in alfalfa, the advantages of this invention are:

[0048] This invention marks the first use of CRISPR / Cas9 technology in the cross-pollinated, autotetraploid plant alfalfa, resulting in homozygous mutant plants. It also represents the first use of the MtU6 promoter to initiate sgRNA transcription in alfalfa. The MtU6 promoter, derived from a closely related species of alfalfa, *Alfalfa trichomoniasis*, has been shown to initiate snRNA (small nuclear RNA) transcription in *Alfalfa trichomoniasis*, achieving gene expression silencing in *Alfalfa trichomoniasis* through RNAi and CRISPR / Cas9. Using *Alfalfa trichomoniasis* MtU6 to initiate sgRNA transcription in alfalfa is more efficient than using other more distantly related species.

[0049] 1. This invention enables the production of hymenoplasts of cross-pollinated alfalfa within one generation.

[0050] Homozygous mutants have significant scientific research and application value and can accelerate alfalfa breeding.

[0051] 2. For the first time, a CRISPR / Cas9 gene knockout platform based on Agrobacterium tumefaciens transformation was established in alfalfa, laying the foundation for gene editing in alfalfa and other plants. It can directly mutate on the gene coding sequence to silence and lose the function of the target gene. This technology is more thorough and stable.

[0052] 3. Compared with natural mutations and traditional artificially induced mutations, this invention introduces mutations with higher mutation efficiency and can achieve a mutation rate of 52% (of which the homozygous mutation rate reaches 12%).

[0053] 4. This technology opens up a new field for gene editing in alfalfa, laying the technical foundation for subsequent simultaneous silencing of multiple genes, deletion of large chromosome segments, and precise insertion of exogenous genes into target sites. At the same time, by utilizing the site-directed function of CRISPR / Cas9, we can mutate and inactivate the Cas9 protein to become dCas9, and achieve upregulation / downregulation, methylation / demethylation, and base substitution of target genes by coupling other functional protein domains to dCas9. Attached Figure Description

[0054] Figure 1 shows the structure of the MsCRISPR / Cas9 expression vector;

[0055] Figure 2 shows wild-type and mutant regenerated plants obtained by infecting alfalfa with the CRISPR / Cas9 expression vector MsCRISPR / Cas9::PDS containing the PDS gene target through Agrobacterium tumefaciens infection.

[0056] Figure 3 shows the sequencing results of PDS gene PCR products from wild-type and mutant alfalfa plants;

[0057] Figure 4 shows the agarose gel electrophoresis results of the regenerated plants obtained by infecting alfalfa with Agrobacterium tumefaciens containing the CRISPR / Cas9 expression vector targeting the PALM1 gene, and the screening of mutant plants by PCR-RE detection.

[0058] Figure 5 shows the leaf phenotypes of wild-type and mutant plants obtained by infecting alfalfa with Agrobacterium tumefaciens containing the CRISPR / Cas9 expression vector MsCRISPR / Cas9::PALM1, which targets the PALM1 gene.

[0059] Figure 6 shows the sequencing results of PALM1 gene PCR products from wild-type and mutant alfalfa plants. Detailed Implementation

[0060] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following technical solutions.

[0061] Example 1: Mutation of the PDS gene encoding phytoene dehydrogenase in alfalfa using the CRISPR / Cas9 system

[0062] Phytoene desaturase (PDS) is the main rate-limiting enzyme in the carotenoid pigment synthesis pathway. It catalyzes the synthesis of colorless C40 phytoene into ζ-carotene, streptolysin, lycopene, 3,4-dehydrolycopene, 3,4,3'4'-dehydrolycopene, or 3,4-dehydrostreptolysin. Mutants of this gene exhibit an albino phenotype, which is easy to observe. Using this gene as a target gene in plants is a convenient method to detect whether a gene knockout system is working and to assess its efficiency.

[0063] Based on the PDS sequence in *Alfalfa*, a close relative of alfalfa, primers were designed to amplify and sequence the *Alfalfa* PDS gene. Target sites were designed based on the obtained PDS gene sequence and referencing cases in *Alfalfa*. The target sites were ligated to the site of vector 1300DM-MtU6(Aar1)-Cas9 to obtain a PDS gene knockout vector. This vector was then used to transform alfalfa explants using the *Agrobacterium tumefaciens* transformation method described above, resulting in regenerated plants (Figure 2). The process of expression vector construction, *Agrobacterium* strain preparation, transformation using *Agrobacterium*, and mutant screening and detection is as follows:

[0064] I. The process of constructing the expression carrier is as follows:

[0065] 1. Design a pair of oligo primers (18-24 bp) based on the target sequence to form the following DNA fragment with adapters at both ends, and synthesize it:

[0066] 5'-TTTG N16-23-3'

[0067] 3'-C N16-23C AA A-5';

[0068] 2. Anneal the synthesized paired primers as described above;

[0069] 3. The vector 1300DM-MtU6(Aar1)-Cas9 was digested with Aar1 enzyme; after CIP reaction, it was separated in 0.8-1.2% agarose gel, and the linear vector was recovered by gel cutting;

[0070] 4. The annealed phosphorylated DNA fragments were ligated with the gel-recovered 1300DM-MtU6(Aar1)-Cas9 linear vector using T4 DNA ligase in a 16°C water bath for 10–16 h.

[0071] 5. The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method. After recovery, the cells were plated on LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12-16 h.

[0072] 6. Pick a single colony and place it in 1.5 ml of LB liquid medium containing 50 mg / L kanamycin, and shake on a shaker at 37°C for 12-16 h;

[0073] 7. Sequencing of the single-clone bacterial culture was performed using sequencing primers. The correctly ligated clone was placed in 50-100 ml of LB broth containing 50 mg / L kanamycin and incubated at 37°C for 12-16 hours. Plasmid extraction was then performed. The sequencing primers are as follows:

[0074] MtU6-TF:GGCATGCAAGCTTATCGATAC.

[0075] II. Preparation of Agrobacterium strains

[0076] The above expression vector was transformed into competent Agrobacterium tumefaciens strain EHA105 cells by electroporation. After recovery, the cells were plated in YM medium (supplemented with 50 mg / L kanamycin and 250 mg / L rifampin) and cultured at 28°C for 24-48 hours. Single colonies were picked and inoculated into 30-50 ml of YM liquid medium (supplemented with 50 mg / L kanamycin and 250 mg / L rifampin) and shaken at 200 rpm on a shaker at 28°C for 24-48 hours.

[0077] III. Transformation using Agrobacterium tumefaciens

[0078] The DNA sequences expressing the CRISPR / Cas9 system and the Hpt protein (anti-plant selection agent) were integrated into the alfalfa genome using Agrobacterium-mediated transformation. This enabled the expression products of these elements to function, and positive plants with silenced target genes were obtained through screening. The specific transformation method is as follows:

[0079] 1. Alfalfa seed germination: Select plump, round alfalfa seeds. Soak in 75% alcohol for 2 minutes, then wash twice with sterile water for 1 minute each time; soak in 0.1% mercuric chloride and shake by hand for 10 minutes, then wash five times with sterile water. Spread the sterilized seeds in sterile petri dishes containing filter paper and air dry. Inoculate onto MS solid medium and germinate in a light incubator or culture room for 7-14 days.

[0080] 2. Callus induction: Cotyledons and hypocotyls of the above-mentioned germinating seedlings were cut into small pieces with a sterile scalpel and inoculated into callus induction medium. The pieces were then cultured in the dark at 25±1℃ in an incubator or culture room for 3 days. The callus induction medium consisted of: SH basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 0.3 mg / L hydrolyzed complexin + 30 g / L sucrose + 8 g / L agar.

[0081] 3. Infection: 1-2 days in advance, inoculate the prepared Agrobacterium tumefaciens strain into 50-100 ml of YM liquid medium and incubate at 28°C on a shaker at 200 rpm until the OD value (260 / 280) is between 0.5-0.8. Transfer the bacterial culture to a 50 ml sterile centrifuge tube and centrifuge at 4000 rpm for 12 minutes at 4°C. Remove the centrifuge tube, discard the supernatant, and resuspend the culture in resuspension until the OD value (260 / 280) is between 0.5-0.8. Add 100 μmol of acetylsuccinyl syringone per liter of culture medium, corresponding to the volume of 100 μmol / L of acetylsuccinyl syringone. Acetyleugenol / ml was used as the resuspension medium in MS liquid medium (MS + 30 g / L sucrose). The exogenous bodies induced in step 2 were collected into a 100 ml sterile Erlenmeyer flask with a breathable plastic sealing film. An appropriate amount of the resuspension solution was poured in (enough to cover the material). The flask was sealed with the sealing film provided with it. The vacuum pump was used to evacuate the flask to 0.5 kPa for 1 hour, with a gentle shake every 15 minutes. The Erlenmeyer flask was then removed and shaken on a shaker at 28°C at 120 rpm for 1 hour. The bacterial solution was poured off and the material was spread out on a sterile Petri dish lined with filter paper to air dry.

[0082] 4. Co-culture: Inoculate the dried, infected material onto a co-culture medium (with a sterile filter paper placed in the medium). The co-culture medium consists of: MS basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 30 g / L sucrose + 8 g / L agar + 100 μmol / L acetylsylgenone. Incubate in the dark at 25 ± 1℃ in an incubator or culture room for 3 days.

[0083] 5. Screening: The co-cultured materials were inoculated onto the screening medium, which consisted of: SH basal medium + 2 mg / L 2,4-D + 0.2 mg / L KT + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 250 mg / L carbenicillin + 15 mg / L hygromycin; cultured under light in an incubator or culture room at 25±1℃ for 30-60 days.

[0084] 6. Differentiation: The recovered material was transferred to differentiation medium, which consisted of: UM basal medium + 2 g / L hydrolyzed complexin + 0.4 mg / L kinetin + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 5 mg / L hygromycin.

[0085] 7. Rooting: Transfer the 1-3cm differentiated buds to the rooting medium, which consists of: MS basal medium + 1 mg / L indolebutyric acid + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime.

[0086] IV. Screening and Detection of Mutants

[0087] Target site deep sequencing detection: DNA samples were extracted from the obtained regenerated plants and amplified using specific amplification primers targeting the PDS sequence of the target gene. The PCR product was TA cloned and ligated into the pMD19-T vector, transformed into E. coli strain DH5α, and a batch of single clones were selected for target site deep sequencing to analyze whether there were mutations at the target site and to analyze the mutation type.

[0088] Through experiments and testing, we found that:

[0089] Phenotypic observation yielded an albino seedling (Figure 2). DNA samples were extracted from the albino seedling and the PDS gene sequence was amplified. Sequencing results (Figure 3) showed that the plant had a 2bp deletion at the target site, which is the cleavage site of the Cas9 protein.

[0090] Figure 2 shows the wild-type (PDS-wt) and mutant (PDS-mt) plants. Wild-type plants exhibited a normal green phenotype, while mutant plants exhibited a white phenotype. This result is consistent with the phenotype of plants obtained through CRISPR / Cas9-mediated PDS gene knockout in species such as poplar and apple. This suggests that the resulting albino seedlings are likely caused by PDS gene mutations mediated by the introduced CRISPR / Cas9 system.

[0091] Figure 3 shows that, based on sequencing results, the PDS mutant plants, compared to the wild type, have a 2bp deletion at the target site, and this mutation occurs at the cleavage site of the Cas9 protein. This indicates that the alfalfa albino seedlings in Figure 2 are caused by CRISPR / Cas9 cleaving the PDS gene target site, resulting in a 2bp deletion during gene repair.

[0092] Example 2: PALM1 gene mutation in alfalfa

[0093] The difference from Example 1 is that:

[0094] Based on the PALM1 sequence in alfalfa's close relative, *Alfalfa tribulus*, primers were designed to amplify and sequence the *MsPALM1* gene sequence of alfalfa. Target sites were designed based on the obtained *MsPALM1* gene sequence. The target sites were ligated to the site of the vector *MsCRISPR / Cas9* to obtain the knockout vector *MsCRISPR / Cas9::PALM1* targeting the *MsPALM1* gene. This vector was transformed into alfalfa explants using the *Agrobacterium tumefaciens* transformation method described above, and regenerated plants were obtained. The detection method was PCR-RE. Genomic DNA samples were extracted from the obtained regenerated plants and amplified using specific amplification primers containing the target sequence of the *MsPALM1* gene. Mutant strains were screened by digesting the PCR products with BstUI restriction endonuclease (Figure 4). The mutation was confirmed by phenotypic observation (Figure 5) and target site deep sequencing (Figure 6).

[0095] Figure 4 shows that, through PCR-RE detection, among the 25 alfalfa regenerated plants sampled, there were 13 mutant plants (with a band at 768 bp that cannot be cleaved by the restriction endonuclease BstUI), of which 10 were heterozygous mutants (in addition to the band at 768 bp that cannot be cleaved by the restriction endonuclease BstUI, there were also two bands generated by the restriction endonuclease BstUI, located at 442 bp and 326 bp, respectively), and 3 were homozygous mutants (with only a band at 768 bp that cannot be cleaved by the restriction endonuclease BstUI); the mutation rate reached 52%, of which the homozygous mutation rate reached 12%.

[0096] Figure 5 shows that among the 25 alfalfa regenerated plants sampled, three plants (numbers 2, 3, and 4 from left to right) exhibited significant phenotypic changes in their compound leaves. Compared to the control (number 1 from left to right), the mutant plants had five leaflets instead of three. Mutant number 3 (number 20 in Figure 4) showed a weak band that could be cleaved by the BstUI restriction endonuclease in the PCR-RE results, which may be because the T0 generation plants are chimeras.

[0097] Figure 6 shows that a portion of the plants selected from Figure 4 were subjected to deep sequencing targeting the MsPALM1 gene, and mutations were successfully introduced at the predetermined target site of CRISPR / Cas9 on the MsPALM1 gene.

[0098] The above results demonstrate that site-directed mutagenesis was successfully achieved in alfalfa using CRISPR / Cas9 technology.

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Claims

1. A method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system, characterized in that, The method includes the following steps: Step (1) Construct MsCRISPR / Cas9, a universal binary expression vector for alfalfa mediated by Agrobacterium tumefaciens; Step (2) Design a CRISPR / Cas9-based target sequence for the target gene in alfalfa, and connect the DNA fragment containing the target sequence to the universal alfalfa vector MsCRISPR / Cas9 to construct the vector MsCRISPR / Cas9::target; Step (3) uses Agrobacterium tumefaciens to mediate the transformation of alfalfa, thereby achieving site-directed mutation of specific genes in alfalfa.

2. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system according to claim 1, characterized in that, The alfalfa mentioned includes wild varieties, local varieties, bred varieties, and introduced varieties.

3. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system according to claim 1, characterized in that, The target gene includes any biologically functional gene or DNA sequence in the alfalfa genome.

4. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system according to claim 1, characterized in that, The full sequence of the binary expression vector MsCRISPR / Cas9 for expressing the CRISPR / Cas9 system in alfalfa is shown in SEQ ID NO.

1.

5. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system according to claim 1, characterized in that, The binary expression vector MsCRISPR / Cas9 for expressing the CRISPR / Cas9 system in alfalfa is a pCambia1300 vector. The pCambia1300 vector contains a T-DNA region for plant transformation. This T-DNA region includes a left-bound repeat (LB repeat), a right-bound repeat (RB repeat), and transformation sequences within the left and right boundaries. The transformation sequences within the left and right boundaries of the T-DNA region include an Hpt gene expression element expressing the protein against the plant selection agent hygromycin, an expression element expressing the Cas9 protein, an expression element expressing sgRNA, and a target sequence between two Aar1 restriction sites within the sgRNA expression element. The Hpt gene expression element contains a Camv 35S promoter-enhanced promoter sequence to initiate Hpt gene transcription, a CDS sequence expressing the Hpt gene, and a Camv poly A termination sequence to terminate Hpt gene transcription. The Cas9 protein expression element contains a 2xCaMV 35S sequence to initiate Cas9 sequence transcription. The expression element expressing sgRNA contains a promoter sequence, a DNA sequence expressing Cas9, and a Nos terminator sequence terminating Cas9 transcription. The expression element expressing sgRNA contains an alfalfa MtU6 promoter sequence that initiates transcription of the sgRNA sequence and a DNA sequence containing the sgRNA.

6. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in claim 5, characterized in that, The sequence of the MtU6 promoter is shown in SEQ ID NO.

2.

7. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in claim 5, characterized in that, The DNA sequence of the expressed sgRNA is shown in SEQ ID NO.

3. The DNA sequence from position 1 to position 30 contains two AarI restriction endonuclease recognition sites, which are used to construct a site-directed mutagenesis expression vector for the target gene.

8. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in any one of claims 1-7, characterized in that, The method for constructing the expression vector MsCRISPR / Cas9::target is as follows: Step (1) Design a pair of 18-24bp oligo primers based on the target sequence to form the following DNA fragment with adapters at both ends, and synthesize it: 5’-T T T G N 16-23- 3’ 3’-C N 16-23 C A A A-5’; Step (2) The synthesized primer pair is annealed to form a complementary oligo primer dimer double-stranded fragment with sticky ends. Step (3) The binary expression vector MsCRISPR / Cas9 was digested with AarI restriction endonuclease; after dephosphorylation by bovine alkaline phosphatase CIP, it was separated by electrophoresis on a 0.8-1.2% agarose gel and the linear vector was recovered and purified. Step (4) The complementary double-stranded DNA fragment with sticky ends after annealing and phosphorylation is ligated with the recovered and purified MsCRISPR / Cas9 linear vector in a 16°C water bath for 10-16 h using T4 DNA ligase. Step (5) The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method. After recovery, the cells were plated on LB agar plates containing 50 mg / L kanamycin and incubated at 37°C for 12-16 h. Step (6) Pick a single colony clone and place it in 1.5 ml of LB liquid medium containing 50 mg / L kanamycin, and shake it on a shaker at 37°C for 12-16 h; Step (7) Sequencing of the single-clone bacterial culture was performed using sequencing primer MtU6-TF. The correct single clone was selected and ligated into 50-100 ml of LB liquid medium containing 50 mg / L kanamycin. The culture was shaken at 37°C for 12-16 h, and the plasmid was extracted.

9. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in claim 6, characterized in that, The sequence of the sequencing primer MtU6-TF is shown in SEQ ID NO.

4.

10. The method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in claim 1, characterized in that, The method for obtaining mutant plants from alfalfa through Agrobacterium tumefaciens-mediated transformation includes the following steps: Step (1) Recovery of Agrobacterium tumefaciens strain: The Agrobacterium strain transformed by electroporation was spread on YM solid plate medium and cultured at 25-29℃ for 24-48h; single colonies were picked and inoculated into 30-50ml YM liquid medium and shaken on a shaker at 100-220r / min at 25-29℃ for 24-48h; both YM solid medium and YM liquid medium contained 30-60mg / L kanamycin and 200-450mg / L rifampin; Step (2) Obtaining alfalfa callus: The process of obtaining alfalfa callus is as follows: Select plump, round alfalfa seeds, soak them in 65-80% alcohol for 1-3 minutes, wash them with sterile water 2-4 times, 0.5-2 minutes each time; soak them in 0.05-0.15% mercuric chloride and shake them by hand for 6-12 minutes, wash them with sterile water 3-5 times, inoculate them on MS solid medium, and germinate them in a light incubator or culture room for 7-16 days; Take the germinating cotyledons and hypocotyls, cut them into small pieces, and inoculate them into callus induction medium. Incubate in the dark at 25±1℃ for 2-4 days. The callus induction medium consists of: SH basal medium + 1.0-3.0 mg / L 2,4-difluorophenoxyacetic acid + 0.1-0.3 mg / L kinetin + 0.1-0.5 mg / L hydrolyzed complexin + 15-40 g / L sucrose + 5.8-9 g / L agar. Preferably, the callus induction medium consists of: SH basal medium + 2 mg / L 2,4-difluorophenoxyacetic acid + 0.2 mg / L kinetin + 0.3 mg / L hydrolyzed complexin + 30 g / L sucrose + 8 g / L agar. Step (3) Agrobacterium tumefaciens infection of alfalfa callus: The process of Agrobacterium tumefaciens infection of alfalfa callus is as follows: 1-2 days in advance, the Agrobacterium tumefaciens strain revived in step (1) is inoculated into 50-100 mL of YM liquid medium and cultured on a shaker at 25-29℃ at 100-220 r / min until the OD value of 260 / 280 is between 0.5-0.8; the bacterial solution is transferred to a 50 mL sterile centrifuge tube and centrifuged at 0-8℃ at 3000-4500 r / min for 10-15 minutes. Discard the supernatant, add resuspension solution and resuspend until the OD value of 260 / 280 is between 0.5 and 0.8, add 50-150 μmol / L acetylsuccinone; the resuspension solution is MS basal medium + 30 g / L sucrose; collect the callus induced in step (2) into a sterile Erlenmeyer flask with a breathable plastic sealing film, pour in the resuspended Agrobacterium tumefaciens solution, seal with the sealing film provided with the Erlenmeyer flask, evacuate to 0.5 kPa in a vacuum pump for a total of 0.5-1.5 h; take out the Erlenmeyer flask, shake on a shaker at 100-150 r / min at 25-29℃ for 0.5-1.5 h, pour out the dry bacterial solution, and air dry; Step (4) Co-culture alfalfa callus infected with Agrobacterium tumefaciens: The co-culture process is as follows: The dried infected material is inoculated into the co-culture medium, a sterilized filter paper is placed in the medium, and the medium is incubated in the dark at 25±1℃ for 2-5 days; The composition of the co-culture medium is: MS basal medium + 1-3 mg / L 2,4-difluorophenoxyacetic acid + 0.1-0.3 mg / L kinetin + 15-40 g / L sucrose + 5.8-9 g / L agar + 50-150 μmol / L acetosyringone; Preferably, the composition of the co-culture medium is: MS basal medium + 2 mg / L 2,4-difluorophenoxyacetic acid + 0.2 mg / L kinetin + 30 g / L sucrose + 8 g / L agar + 100 μmol / L acetosyringone; Step (5) Screening and culture of alfalfa callus: The screening and culture process is as follows: the co-cultured material is inoculated into the screening medium and cultured under light in an incubator or culture room at 25±1℃ for 30-60 days; the screening medium consists of: SH basal medium + 1-3 mg / L 2,4-difluorophenoxyacetic acid + 0.1-0.3 mg / L kinetin + 15-40 g / L sucrose + 5.8-9 g / L agar + 150-450 mg / L cefotaxime + 150-450 mg / L carbenicillin + 10-50 mg / L hygromycin; preferably, the screening medium consists of SH basal medium + 2 mg / L 2,4-difluorophenoxyacetic acid + 0.2 mg / L kinetin + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 250 mg / L carbenicillin + 15 mg / L hygromycin; Step (6) Alfalfa callus differentiation culture: The screened and cultured materials are transferred to differentiation medium and cultured under light in an incubator or culture room at 25±1℃ for 15-30 days; the differentiation medium consists of: UM basal medium + 0.5-5 g / L hydrolyzed complexin + 0.1-2 mg / L kinetin + 15-40 g / L sucrose + 5.8-9 g / L agar + 150-450 mg / L cefotaxime + 4-10 mg / L hygromycin; preferably, the differentiation medium consists of: UM basal medium + 2 g / L hydrolyzed complexin + 0.4 mg / L kinetin + 30 g / L sucrose + 8 g / L agar + 250 mg / L cefotaxime + 5 mg / L hygromycin; Step (7) Rooting culture of alfalfa regenerated shoots: The differentiated 1-3cm shoots are transferred to the rooting medium, which consists of: MS basal medium + 0.5-2mg / L indolebutyric acid + 15-40g / L sucrose + 5.8-9g / L agar + 150-450mg / L cefotaxime; preferably, the rooting medium consists of: MS basal medium + 1mg / L indolebutyric acid + 30g / L sucrose + 8g / L agar + 250mg / L cefotaxime; Step (8) Screening and detection of alfalfa mutants, wherein the methods for screening and detecting alfalfa mutants include PCR-RE detection, T7E1 enzyme digestion detection and target deep sequencing.

11. The application of the method for site-directed mutagenesis of alfalfa genes using the CRISPR / Cas9 system as described in any one of claims 1-10 in alfalfa breeding and gene-edited alfalfa breeding.