Plant gene editing system and method for delivering CRISPR / Cas9 based on mobile RNA

Abstract

The invention discloses a plant gene editing system and method for delivering CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 based on mobile RNA (ribonucleic acid). The system does not depend on tissue regeneration or protoplast culture, Cas9 and gRNA are remotely delivered from the root to enter the overground part by fusing a CRISPR / Cas9 gene editing carrier of a tRNA-like sequence (TLS) movable motif and combining a hairy root transformation system, target gene editing is completed, and heritable gene editing seeds can be obtained at the current generation. The method comprises the following steps: 1, constructing a movable CRISPR / Cas9 gene editing fusion vector, wherein the movable CRISPR / Cas9 gene editing fusion vector comprises a target vector, an intermediate vector and an editing vector; and 2, application of the agrobacterium rhizogenes mediated plant hairy root transformation system. And 3, detecting the mobility and editing type of the movable editing carrier. And 4, gene function verification application. The system greatly breaks through the bottlenecks of difficult plant genetic transformation and long time consumption of precise breeding, and provides theoretical basis and technical support for gene function research and germplasm innovation of important agronomic traits of plants.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to a plant gene editing system and method based on mobile RNA delivery of CRISPR / Cas9. Background Technology

[0002] In the post-genomic era, with the rapid development of high-throughput sequencing, crop domestication, the mining of genomes of important agronomic traits, and multi-omics joint analysis play a crucial role in guiding precision crop breeding. In recent years, the publication of high-quality genomic data from various crops has highlighted the growing demand for research on key gene resources for important agronomic traits and the elucidation of their functional mechanisms. However, many crops, such as heading cabbage, radish, and maize, currently suffer from low genetic transformation efficiency, regeneration difficulties, and genotype dependence, lacking efficient and stable genetic transformation technologies. This severely restricts the improvement of superior crop traits and the progress of germplasm innovation.

[0003] To address the above issues, it is particularly important to develop a highly efficient, stable crop gene editing system that does not rely on stable genetic transformation. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies and break through the bottlenecks of difficult crop genetic transformation, time-consuming precision breeding, and exogenous gene elimination in offspring, this invention provides a CRISPR / Cas9 gene editing vector incorporating a TLS mobile motif. This system enables long-distance delivery... Cas9 and gRNA Components. Combined with the Agrobacterium rhizogenes-mediated plant hairy root transformation system, it is possible to obtain components with expression... Cas9 -TLS / gRNA -TLS transgenic hairy roots, when Cas9 and gRNA Once delivered to the reproductive organs, the gene-edited components function to produce heritable, non-GMO offspring in the present generation.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution: 1. Construction of a mobile CRISPR / Cas9 gene editing fusion vector, including the following: S1. Construction of a portable CRISPR / Cas9 target vector S2. Construction of a mobile CRISPR / Cas9 intermediate vector Construction of S3 and a mobile CRISPR / Cas9 gene editing vector 2. Application of Agrobacterium rhizogenes-mediated plant hairy root transformation system 3. Testing the mobility and editing effect of the movable editing platform 4. Applications in gene function verification Attached Figure Description

[0006] Figure 1A pMDC7+rbcs-e9t terminator vector construction process.

[0007] Figure 1B pMDC7+Cas9Null+rbcs-e9t terminator vector construction process.

[0008] Figure 1C pMDC7+Cas9tMet+rbcs-e9t terminator carrier construction process.

[0009] Figure 1D pMDC7+Cas9tMetdDt+rbcs-e9t terminator carrier construction process.

[0010] Figure 2A Flowchart of the construction process of intermediate carrier pENTR4+tMet+gNIA1tMet.

[0011] Figure 2B Flowchart of the construction process of intermediate carrier pENTR4+tMetdDt+gNIA1tMetdDt.

[0012] Figure 2C Flowchart for constructing intermediate carrier pENTR4+Null+gNIA.

[0013] Figure 3 Intermediate vector transcription unit.

[0014] Figure 3A Schematic diagram of the construction of the :pMDC7+Cas9tMet+rbcS-e9t terminator+gNIA1tMet vector.

[0015] Figure 3B Schematic diagram of vector construction: pMDC7+Cas9tMetdDT+rbcS-e9t terminator+gNIA1tMetdDT

[0016] Figure 3C Schematic diagram of vector construction: pMDC7+Cas9Null+rbcS-e9t terminator+gNIA1Null.

[0017] Figure 4A PCR detection results of Cas9 transformed seedling root DNA using a mobile gene-editing vector.

[0018] Figure 4BRT-PCR results of Cas9 transformed into seedling root RNA using a mobile gene editing vector.

[0019] Figure 5A Mobility testing of NIA1 transformed seedlings.

[0020] Figure 5B Mobility testing of PPR-transformed seedlings.

[0021] Figure 6 PPR transformation seedling editing and testing.

[0022] Figure 7A PPR-transformed seedlings with dual gRNA editing type.

[0023] Figure 7B The single gRNA editing type in the second copy of the NIA1 gene in NIA1 transformed seedlings.

[0024] Figure 8 Statistics on the editing efficiency of tissue samples from NIA1 transformed seedlings.

[0025] Figure 9 Wild-type A03 (left) and NIA1 transformed seedlings (right).

[0026] Figure 10 Screening of NIA1 transformed seedlings for generation T1 using ammonium-deficient nitrogen culture medium.

[0027] Figure 11 Molecular validation of T1 generation.

[0028] The mobile CRISPR / Cas9 gene editing technology system provided by this invention can be applied to other implementation schemes, as well as modified schemes, improved schemes, equivalent schemes, and other technical solutions that do not depart from the spirit and scope of this invention. The following description is only for example; Chinese cabbage A03 is just a material commonly used by our research group and is not considered a specific material.

[0029] The mobile CRISPR / Cas9 gene editing fusion vector described in 1 is constructed, wherein the target vector is characterized in that: pMDC7 is an estradiol-induced binary vector, and step S1 includes the following steps: S1-1. Select two restriction enzyme sites, XhoⅠ and AscⅠ, to linearize the pMDC7 vector. The double digestion system is shown in Table 1, and the vector sequence is shown in Appendix SEQ ID NO.1. Incubate overnight at 37℃, and recover the target fragment of the correct size using gel extraction.

[0030] S1-2. Using pJF1031 as a template, specific primers were used to amplify the rbcs-e9t terminator (663bp), Cas9Null (4295bp), Cas9tMet (4349bp), and Cas9tMetdDt (4321bp) fragments. The high-fidelity enzyme KOD-Plus-Neo was used for amplification. The PCR amplification system is shown in Table 2. The pJF1031 sequence is shown in Appendix SEQ ID NO.2, and the primer sequences are shown in Appendix Table A8. The PCR products were gel-cleaved, purified, and then double-digested with XhoⅠ and AscⅠ. The reaction system was the same as in Table 1, and the digested products were recovered from the gel.

[0031] S1-3. The target fragment and the linearized pMDC7 vector were recombined using T4 ligase, respectively. The mixture was incubated overnight at 16°C and the reaction was terminated by incubation at 65°C for 10 min. The reaction system is shown in Table 3 below.

[0032] S1-4 and the ligation products were transformed into DB3.1 competent cells. After overnight culture, single clones were picked and identified by colony PCR. Positive clones were selected, and the target vector, including pMDC7+rbcs-e9t terminator, was obtained after successful sequencing. Figure 1A ), pMDC7+Cas9Null+rbcs-e9t terminator( Figure 1B ), pMDC7+Cas9tMet+rbcs-e9terminator( Figure 1C ) and pMDC7+Cas9tMetdDt+rbcs-e9t terminator( Figure 1D ), where Cas9Null does not carry a mobile element, while Cas9tMet and Cas9tMetdDt carry tRNA respectively. Met tRNA Met Moving components such as dDT.

[0033] Table 1. Double digestion system of pMDC7 vector and rbcs-e9t terminator fragment

[0034] Table 2 KOD PLUS NEO enzyme (high-fidelity enzyme) reaction system

[0035] Table 3. pMDC7+rbcs-e9t terminator vector T4 ligase reaction system

[0036] The intermediate vector, constructed according to the mobile CRISPR / Cas9 gene editing fusion vector described in 1, is characterized in that its template requires fusion with two... gRNA , gRNA1 and gRNA2 Step S2 includes the following steps: S2-1, using F2-Null, F2-tMet, and F2-tMetdDt as templates, and two target genes... gRNA Using primers containing the primer and adapter sequence, F2 fragments carrying different motifs were amplified. Specific primer and adapter sequences are shown in Table 4.

[0037] Table 4. Specific primer and adapter sequences

[0038] Note: Underlined sequence: enzyme recognition site; Green sequence: gRNA Sequence; Blue sequence: gRNA scaffold; gray sequence: pU6-29.

[0039] We selected two target genes (a) and (b) for editing. We used g BraNIA1 FP / g BraNIA1 RP and g BraPPR FP / g BraPPR RP primers were used to amplify F2 fragments carrying different motifs. Primer sequences are shown in Appendix A8, and F2 template sequences with different motifs are shown in Table 5.

[0040] yes BraNIA1 This is a minor cytoplasmic isoform of nitrate reductase (NR). It is the rate-limiting enzyme in the first step of nitrate assimilation.

[0041] BraPPR It encodes a pentapeptide repeat sequence protein that may play a crucial role in mitochondria or chloroplasts by binding to organelle transcripts and is associated with leaf color formation.

[0042] We used the online website (ACEofBASEs https: / / aceofbases.cos.uni-heidelberg.de:8044 / ) to select target sites. On the ACEofBASEs website, we entered... BraNIA1 and BraPPR For the gene, select *Brassica napus* Ensembl Plants V 45 from the species. After submission, select target sequences, initially choosing two target sites for each gene. NIA1 The gene has three copies in Chinese cabbage A03, which will NIA1The three copies were sequence aligned using the snapgene software. Target sequences were then screened based on the homologous sequences of these three copies. The selected target sequences are shown in Table 6.

[0043] Table 5 F2 fragment sequences

[0044] Note: Brown sequence: tU6-26; Blue sequence: s gRNA scaffold; Green sequence: pU6-29; Red sequence: TLS Table 6 BraNIA1 and BraPPR target sequence

[0045] S2-2. After gel recovery and purification of the PCR amplification product, it was digested with BsaI to generate sticky ends for the target fragment. The digestion reaction system is shown in Table 7.

[0046] S2-3, the backbone vectors pENTR4+Null, pENTR4+tMet, and pENTR4+tMetdDt were double-digested using double BsaI restriction sites to linearize the vectors. The mixtures were incubated overnight at 37°C and then heat-inactivated at 80°C for 20 min. The digestion products were recovered from the gel. The digestion reaction system is shown in Table 7.

[0047] Table 7. Double digestion system of pEntr4 series vectors and F2 fragment

[0048] S2-4, with a purpose gRNA The F2 fragment was recombined with pENTR4+Null, pENTR4+tMet, and pENTR4+tMetdDt, respectively. Ligation was performed overnight at 16°C using T4 ligase, and the reaction was terminated by incubation at 65°C for 10 min.

[0049] S2-5, 10 μl of the ligation product was transduced into TOP10 competent cells. After overnight culture, single clones were collected for colony PCR identification. Positive clones were selected, and after successful sequencing, cells carrying double colonies were obtained. gRNA The intermediate vectors for pENTR4+Null, pENTR4+tMet, and pENTR4+tMetdDt are constructed as shown in Figure 2. The vector transcription units are as follows: Figure 3 .

[0050] The gene editing vector constructed according to the mobile CRISPR / Cas9 gene editing fusion vector described in 1 is characterized by: […]. Cas9-TLS Components and gRNA-TLSThe components are assembled together to form the final mobile CRISPR / Cas9 gene editing vector. Step S3 includes the following steps: S3-1. The intermediate vector and the target vector carry TLS motifs in a one-to-one correspondence and undergo LR reactions. The pMDC7+Cas9Null+rbcs-e9t terminator vector and the pENTR4+Null+F2 vector, the pMDC7+Cas9tMet+rbcs-e9tterminator vector and the pENTR4+tMet+F2 vector, and the pMDC7+Cas9tMetdDt+rbcs-e9t terminator vector and the pENTR4+tMetdDt+F2 vector undergo LR reactions respectively.

[0051] S3-2. It is recommended to add the reaction mixture on ice. First, add all components except LR Clonase II, gently press with a microcentrifuge, and vortex briefly twice to mix thoroughly. Melt the Invitrogen LR Clonase II enzyme mixture on ice for about 2 minutes, vortex the LR Clonase II enzyme mixture briefly twice (2 seconds each time), and add 2 μl to the reaction mixture.

[0052] S3-3, incubate overnight at 25°C. Add 1 µl of proteinase K solution to each sample to terminate the reaction. After thorough mixing, incubate at 37°C for 10 min. The reaction system is shown in Table 8 below.

[0053] S3-4. Transform the above reaction products into TOP10 competent cells, culture overnight, randomly select single clones from the plate, perform colony PCR identification, select positive clones, and obtain tRNA-carrying cells after successful sequencing. Met and tRNA Met The flowcharts for the mobile gene editing vector with the dDT motif and the non-mobile editing vector with the pMDC7+Cas9Null+rbcS-e9t terminator+gNIA1Null motif are shown below. Figure 3 As shown.

[0054] S3-5. Transform all the above vectors into Agrobacterium rhizogenes K599, culture for 48 hours, then randomly select single clones from the plate for colony PCR identification. If the identification is successful, preserve the bacteria for subsequent use.

[0055] Table 8 LR Reaction System

[0056] According to the application of the Agrobacterium rhizogenes-mediated plant hairy root transformation system described in 2, 2 includes the following steps: S1, Hairy Root Transformation Steps S2 and CRISPR / Cas9 mobile editable vector hairy root transformation positive seedlings and transformation efficiency detection According to the application of the Agrobacterium rhizogenes-mediated plant hairy root transformation system described in 2, taking Chinese cabbage A03 as an example, S1 includes the following steps: S1-1. Planting Chinese cabbage: The high-generation homozygous inbred line A03 of Chinese cabbage was provided by the State Key Laboratory of Crop Improvement and Regulation in North China. A03 was germinated for two days and then sown in seedling trays. It was cultured at 25℃ with 12000 Lux and a photoperiod of 16h light / 8h dark for 5 days. At this time, the cotyledons of the Chinese cabbage seedlings were fully unfolded.

[0057] S1-2, Small amount of activated bacterial strain: Add 50 μl of bacterial suspension (K599) to 2 ml of LB medium (corresponding antibiotic for the carrier + 100 μg / ml strep), and revive at 28℃ and 200 rpm for 24 h.

[0058] S1-3. Large-scale activation of bacterial strains and plate inoculation: Take 500 μl of the activated bacterial culture above and add it to 20 ml of LB medium containing the corresponding antibiotic. Recover at 28℃ and 200 rpm for 24 h. Then take 500 μl of the recovered bacterial culture, centrifuge at 5000 rpm for 2 min, discard 400 μl of supernatant, resuspend the remaining bacterial cells, spread them on LB plates containing the corresponding antibiotic, and incubate at 28℃ for 24 h.

[0059] S1-4. Cut the seedling 1-2 cm below the fully expanded cotyledons of the Chinese cabbage seedling, and obliquely cut the hypocotyl of the Chinese cabbage seedling in fresh Agrobacterium rhizogenes solution (OD600≈0.6-1.0).

[0060] S1-5. Use a sterile applicator to scrape bacteria from an LB agar plate, thoroughly spread the bacteria onto the oblique cut surface of the Chinese cabbage seedlings, plant the treated seedlings in vermiculite, and inject the remaining activated bacterial solution around the seedlings.

[0061] S1-6, cultured in the dark for 2 days, while simultaneously watering with Agrobacterium tumefaciens solution daily, followed by normal light cultivation after two days, maintaining high humidity. After 7-10 days, when roots have re-emerged, the seedlings are placed under normal culture conditions (after infection with the strain using the movable editing vector, once the seedlings have grown roots, they are continuously treated with estrogen (5mM) to induce vector expression).

[0062] S1-7. When the plants have grown to 8-10 true leaves, subsequent tests can be conducted on positive seedlings, carrier mobility, and editing effects. Successfully tested plants are placed in a low-temperature vernalization chamber for approximately 40 days of vernalization treatment. After vernalization, the plants are transferred to normal growth conditions and left to bolt, flower, pollinate, and bear fruit for harvest.

[0063] The application of the Agrobacterium rhizogenes-mediated plant hairy root transformation system according to 2 is characterized in that S2 includes the following steps: S2-1. After the transformed plants obtained in S1 have regrowed roots for 7-10 days, root DNA is extracted from the transformed plants and amplified using Cas9Seq FP5 / Cas9 RT-RP (415bp) primers. Cas9 The gene and primer sequences are shown in Appendix A8. This test verifies whether the mobile CRISPR / Cas9 gene editing vector has been successfully transferred into the plant.

[0064] S2-2. Extract RNA from the roots of successfully transformed plants, reverse transcribe cDNA, and amplify using Cas9Seq FP5 / Cas9 RT-RP (415bp) primers. Cas9 Genes were examined to determine whether the mobile CRISPR / Cas9 gene editing vector was expressed.

[0065] S2-3. Calculate the conversion rate according to the formula below.

[0066] Reference formula for calculating the transformation rate of mobile CRISPR / Cas9 gene editing vector-transformed seedlings: Conversion rate = (Number of seedlings with Cas9-positive roots / Total number of converted seedlings) * 100% In the experiment, we tested the hairy root transformation positive seedlings and transformation efficiency of the target genes in (a) and (b), and the results are as follows: Figure 4A As shown in Table 9. The number of tests and conversion rates are shown in Table 9. BraNIA1 A total of 159 gene-edited transformed seedlings were tested, and [the virus] was detected in 69 of them. Cas9 The gene conversion rate was 43.4%. BraPPR A total of 116 gene-edited transformed seedlings were tested, of which 81 were Cas9-positive, resulting in a transformation rate of 68.6%. To further investigate... Cas9 We detect the expression of [something], and we [do something]. NIA1 Transformed seedlings and PPR RT-PCR was performed on root samples from transformed seedlings. The RT-PCR results are shown in the figure. Figure 4B Although the brightness of the bands varied, bands 1 through 12 were all detected. Cas9 The results above demonstrate that the movable editing vector can achieve stable transformation and expression in the hairy root transformation system. Cas9 Expression. For example... Figure 4A : Note: M: DL 500 marker; -: water control; +: positive control; 1-12: seedlings transformed with mobile gene editing vector.

[0067] like Figure 4B : Note: M: DL 500 marker; -: water control; +: positive control; 1, 2: NIA1 Null-converted seedlings, 3, 4: NIA1 tMet conversion seedlings, 5, 6: NIA1 tMetdDt transformed seedlings, 7, 8: PPR Null-transformed seedlings, 9, 10: PPR tMet transformed seedlings, 11, 12: PPR tMetdDt transformed seedlings.

[0068] Table 9 Transformation rate of seedlings transformed with mobile gene-editing vectors

[0069] According to the movable editing carrier mobility and editing type detection described in 3, 3 includes the following: S1. Mobility Detection of Mobile CRISPR / Cas9 Gene Editing Vectors S2, Detection of Editing Type of Mobile CRISPR / Cas9 Gene Editing Vector To achieve the goal of detecting the mobility of the S1 mobile CRISPR / Cas9 gene editing vector, the following steps are included: S1-1. RNA was extracted from the above-ground leaf tissue and underground tissue of positively transformed seedlings. 3-5 leaves were randomly selected from the above-ground tissue of each seedling, and a portion of the root system was randomly selected from the underground tissue of each seedling.

[0070] S1-2. After extracting RNA, reverse transcription is performed to synthesize cDNA.

[0071] S1-3. Using the synthesized cDNA as a template, perform RT-PCR amplification using Cas9 RT-RP / Cas9 RT-FP, gRNA RT-RP / gRNART-FP, Hyg RT-RP / Hyg RT-FP, and Bra-actin-R / Bra-actin-F primers. Cas9 , gRNA , Hyg and Actin The amplification cycle number was 45 to improve sensitivity. The amplification status of underground and aboveground tissues was compared to determine whether migration had occurred. Primer sequences are shown in Appendix A8.

[0072] In the experiment, we performed mobility testing on the target genes in (a) and (b) using a mobile CRISPR / Cas9 gene editing vector. Following the method described in S2, we... NIA1 and PPR RT-PCR was performed on the aboveground leaves of the transformed seedlings. The seedlings carrying TLS elements... Cas9 , gRNA After integration with TLS, it can be detected on the ground, as shown in Figure 5. Hyg As a resistance gene, it is not mobile, does not have TLS elements, and is not detected in the terrestrial portion; Actin As an internal reference gene, it can be detected in both above-ground and below-ground tissues. RT-PCR amplification Cas9 , gRNA , Hyg and Actin The results are as follows Figure 5A ( NIA1 Transformed seedlings). Figure 5B ( PPR The transformed seedlings are shown in the figure. Among them, No. 1 and No. 3 are leaf tissues of seedlings without moving elements, No. 2 and No. 4 are root tissues of seedlings without moving elements, No. 5 and No. 7 are leaves of seedlings with tMet moving elements, No. 6 and No. 8 are roots of seedlings with tMet moving elements, No. 9 and No. 11 are leaves of seedlings with tMetdDt moving elements, and No. 10 and No. 12 are roots of seedlings with tMetdDt moving elements.

[0073] In the examination of aboveground leaf tissue, we selected those that had already been detected in root tissue. Cas9 The samples were tested, and the specific conversion types and mobility are shown in Table 10. NIA1 A total of 9 genetically modified seedlings were produced. NIA1 Four tMet-transformed seedlings were produced. NIA1 Five tMetdDt transformed seedlings were found, including those in... NIA1 Two tMet-transformed seedlings were detected Cas9 ,exist NIA1 Three strains of tMetdDt transformed seedlings were detected Cas9 A total of 5 plants. PPR A total of 7 genetically modified seedlings were produced. PPR Four tMet-transformed seedlings were produced. PPR Three tMetdDt transformed seedlings were found, among which... PPR Three tMet-transformed seedlings were detected Cas9 ,exist PPR Three strains of tMetdDt transformed seedlings were detected Cas9 A total of 6 plants were produced. These two genetically modified seedlings were successfully detected in their above-ground parts. Cas9 The expression.

[0074] Table 10 Mobility Detection of Mobile Gene Editing Vectors

[0075] The above results demonstrate that carrying TLS sequences Cas9 After gene expression, it can migrate and is detected in non-transformed sites, i.e., the aerial parts. For example... Figure 5A : Note: M: DL 500 marker; -: water control; +: positive control; (a): Cas9 Detection; (b): gRNA Detection (c): Hyg Detection; (d): Actin Detection; 1, 3: NIA1 Null-transformed seedling leaves; 2, 4: NIA1 Null-transformed seedling roots; 5, 7: NIA1 tMet-converted seedling leaves; 6, 8: NIA1 tMet transformed seedling roots; 9, 11: NIA1 tMetdDt transformed seedling leaves; 10, 12: NIA1 tMetdDt transforms seedling roots.

[0076] like Figure 5B : Note: M: DL 500 marker; -: water control; +: positive control; (a): Cas9 Detection; (b): gRNA Detection; (c): Hyg Detection; (d): Actin Detection; 1, 3: PPR Null-transformed seedling leaves; 2, 4: PPR Null-transformed seedling roots; 5, 7: PPR tMet-converted seedling leaves; 6, 8: PPR tMet transformed seedling roots; 9, 11: PPR tMetdDt transformed seedling leaves; 10, 12: PPR tMetdDt transforms seedling roots.

[0077] To achieve the purpose of detecting the editing type of the S2 mobile CRISPR / Cas9 gene editing vector, S2 includes the following methods: S2-1, Agarose Gel Electrophoresis Detection S2-2, TA cloning sequencing method S2-3, third-generation amplicon PacBio sequencing The characteristic of S2-1 agarose gel electrophoresis detection is: dual gRNA Meanwhile, gene editing can cause large deletions of genome segments, which are most easily detected by PCR amplification. In the electrophoresis image, we mainly observe whether double... gRNA Short strips caused by editing.

[0078] We studied the target gene (a). NIA1 Transformed seedlings and (b) PPR The modified seedlings underwent edit type testing, respectively from... PPR and NIA1 Genomic DNA was extracted from the underground roots and aboveground parts of the genetically transformed seedlings, and PCR amplification was performed targeting the gene. After analysis, a total of 53 samples were... PPR Among the transformed seedling samples (including underground roots and aboveground parts), 8 samples showed double-edited banding (approximately 350 bp), such as... Figure 6 These results indicate that in the T0 generation transformed seedlings, double... gRNA Editing has been successfully induced at the target site in some plants. For example... Figure 6 : Note: M1: DL 5000 marker; M2: DL 500 marker; -: water control; WT: A03 wild type; 1-6: BraPPR Transformed seedlings The S2-2 TA cloning sequencing method is characterized by its ability to further confirm the sequence of short bands. The S2-2 method includes the following steps: S2-2-1, In gRNA Primers were designed on both sides to amplify the target gene, and the PCR products were recovered by gel extraction.

[0079] S2-2-2. Using the pEASY®-Blunt Zero Cloning Kit, ligate the PCR product to the pEASY®-Blunt Zero Cloning Vector at a ratio of 7:1. Incubate at 25°C. The incubation time depends on the length of the inserted fragment. The reaction system is shown in Table 11 below.

[0080] S2-2-3. The ligation product is added to 50 μL of Trans1-T1 competent cells. After overnight culture, single clones are picked and sent for sequencing. The target site sequence is compared with the wild-type sequence to determine the editing type.

[0081] Table 11 pEASY®-Blunt Zero Cloning Vector Reaction System

[0082] We used NIAe1-FP / NIAe1-1 RP, NIAe1-FP / NIAe1-2 RP, NIAe1-FP / NIAe1-3 RP, and PPREdit-FP / PPR Edit-RP2 primers to amplify the amplification, respectively. BraNIA1 -1, BraNIA1 -2, BraNIA1 -3, BraPPRGene and primer sequences are shown in Appendix Table 8A. After being coupled to the pEASY®-Blunt Zero Cloning Vector, the sample was sequenced, and the sequence was compared with the wild-type sequence to confirm the presence of double gene sequences in the sample. gRNA Mediated editing events. Furthermore, the sequencing results revealed two different types of editing modes, such as... Figure 7A As shown, these mutations conform to the expected double... gRNA Editing effect.

[0083] We continued with 62 samples. NIA1 Editing type detection was performed on the transformed seedlings, but no double editing was found. Considering the single... gRNA In cases where the target sites have been edited, we amplify the full-length gene, ligate it with the pEASY®-Blunt Zero CloningVector vector, and then sequence it to detect sequences near the two target sites. NIA1 A single [item] was found in the second copy. gRNA Editor, such as Figure 7B .

[0084] The characteristic of S2-3 third-generation amplicon PacBio sequencing is that it obtains a full range of edit types.

[0085] We choose NIA1 Copy 1 was subjected to third-generation PacBio amplicon sequencing. From... NIA1 Root systems (detected as Cas9 positive) and leaf tissues from two transformed seedlings were selected, designated R1, R2, L1, and L2, respectively. Floral organ tissues from five seedlings were pooled into two groups, designated F1 and F2. These six samples were sent to the company for third-generation amplicon PacBio sequencing. Results showed that g NIA1 -1 There are a total of 26 editing types, including 13 missing types, 5 replacement types, 8 insertion types, and g NIA1 A total of 24 editing types were identified, including 9 missing types, 5 replacement types, and 10 insertion types. The total editing efficiency was 11.9375% for R1, 11.1111% for R2, 4.6711% for L1, 4.2040% for L2, 3.1156% for F1, and 3.9532% for F2. The average editing efficiency was 11.5213% for root samples, 4.4376% for leaves, and 3.5344% for floral organs. For statistical analysis of editing efficiency among tissue samples, see [link to relevant documentation]. Figure 9 See Appendices A1-A7. The results fully demonstrate the effectiveness of the mobile CRISPR / Cas9 gene editing system in A03.

[0086] According to the gene function verification application described in 4, the characteristic is to detect the effect of the target gene on plants after editing.

[0087] In our experiment BraNIA1 The cytoplasmic minor isoform encoding nitrate reductase is involved in the first step of nitrate assimilation and is the rate-limiting enzyme in the nitrate nitrogen assimilation process. When BraNIA1 Editing disrupts the original gene sequence, leading to errors in gene expression. This prevents the normal translation of nitrate reductase, hindering the utilization of nitrate nitrogen and resulting in weak growth, stunted plants, and chlorotic leaves. To address this... BraNIA1 To verify gene function, we NIA1 Transformed seedlings were continuously induced with estrogen to induce editing of the aboveground parts. The aboveground phenotype was observed. Figure 9 Phenotypic comparison with wild-type A03 revealed... NIA1 The transformed seedlings had lighter leaf color compared to A03, which preliminarily verifies... BraNIA1 The function.

[0088] To verify that the mobile CRISPR / Cas9 gene editing technology system can produce heritable gene-edited seeds in the modern era, we... NIA1 Transformed seedlings underwent vernalization treatment, and seeds were harvested through strict self-pollination. T1 generation seeds were screened using ammonium-deficient MS medium. Figure 10 The preparation method for ammonium-deficient MS medium is as follows: Ammonium-deficient MS medium: Nitrogen-deficient MS medium base salt (containing vitamins) 2256.57 mg, KNO3 505.5 mg, sucrose 30 g, agar powder 8 g, dissolved in 1 L ddH2O, pH adjusted to 5.8, autoclaved at 121℃ for 15 min.

[0089] When plants exhibit weak growth, stunted size, and chlorotic leaves, they are highly likely to be homozygous offspring of the edited plants. We screened 237 plants. NIA1 When the T1 generation of transformed seedlings does not exhibit a similar phenotype after screening on ammonium-deficient MS medium, we need to consider... NIA1 The gene has three copies, and even when a heterozygote is present, no phenotype will appear. Therefore, the T1 generation is... NIA1 Molecular verification of genes ( Figure 11 After amplifying the target gene, ligating it into a T vector, and performing Sanger sequencing, no editing was found. The possible reason is that the T1 generation seed population was too small, therefore the population needs to be expanded for further screening.

[0090] The test reagents and instruments used in the above experiments are shown in Appendix A9 and A10.

[0091] like Figure 11 Molecular validation of T1 generation: Note: M: DL5000 marker; -: water control; +: A03 control; 1-19: monoclonal antibody

[0092] Table A2g NIA1 -1 Insert Editing Type Statistics

[0093] Table A3g NIA1 -1 Replacement Edit Type Statistics

[0094] Table A4g NIA1 -2 Missing Edit Type Statistics

[0095] Table A5g NIA1 -2 Insert Editing Type Statistics

[0096] Table A6g NIA1 -2 Replacement Edit Type Statistics

[0097] Table A7 NIA1 Statistics on average transformation efficiency of transformed seedling tissues

[0098] Table A8 List of primers used in this experiment

[0099] Note: Underlined sequence: restriction enzyme recognition site; lowercase sequence: homologous sequence; green sequence: gRNA Sequence; Blue sequence: gRNA scaffold; gray sequence: pU6-29.

[0100] Table A9 Main Reagents Used in the Experiment

[0101] Table A10 Main Instruments Used in the Experiment

[0102] SEQ ID NO.1 pMDC7 SEQ ID NO.2 pJF1031

Claims

1. A mobile RNA-based CRISPR / Cas9 delivery system and method for plant gene editing, comprising, The method comprises the following steps: The mobile CRISPR / Cas9 gene editing fusion vector construction includes a target vector, an intermediate vector and an editing vector; S1, construction of a mobile CRISPR / Cas9 target vector, comprising the following steps: S1-1, linearization of a pMDC7 vector, selection of an enzyme cutting site, as shown in Table 1, the vector sequence is shown in Appendix SEQ ID NO. 1, 37 DEG C overnight incubation, gel recovery of the target fragment meeting the size; S1-2, specific amplification of rbcs-e9t terminator fragments, Cas9Null fragments and Cas9tMetdDt fragments using pJF1031 as a template, the amplification system is shown in Table 2, the specific primers are shown in Table A8, the pJF1031 sequence is shown in Appendix SEQ ID NO. 2, the PCR product is gel recovered and purified, and then subjected to enzyme cutting using the enzyme cutting site in S1-1, the reaction system is shown in Table 1, and the enzyme cutting product is gel recovered; S1-3, recombination of the target fragments and the linearized pMDC7 vector using T4 ligase, the reaction system is shown in Table 3; S1-4, the connection product is respectively transferred into DB3.1 competent cells, and pMDC7+Cas9tMet+rbcs-e9t terminator9 (FIG. 1A), pMDC7+Cas9Null+rbcs-e9t terminator (FIG. 1B) and pMDC7+Cas9tMetdDt+rbcs-e9t terminator (FIG. 1C) are obtained after successful identification and sequencing, wherein Cas9Null does not carry a mobile element, Cas9tMet and Cas9tMetdDt respectively carry tRNAMet and tRNAMetdDT mobile elements; S2, construction of a mobile CRISPR / Cas9 intermediate vector, characterized in that: the template of the intermediate vector needs to fuse two gRNAs, gRNA1 and gRNA2; and the step S2 comprises the following steps: S2-1, using F2-Null, F2-tMet and F2-tMetdDt as templates, using two gRNA sequences carrying a target gene and a linker sequence as primers, amplifying F2 fragments carrying different TLS, the primer sequence is shown in Appendix A8, the linker sequence is shown in Table 4, and the F2 template sequence is shown in Table 5; S2-2, the PCR amplification product is recovered and purified and then subjected to BsaI enzyme cutting, so that the target fragment is provided with a sticky end; S2-3, double enzyme cutting of double BsaI enzyme cutting sites on the pENTR4+Null, pENTR4+tMet and pENTR4+tMetdDt vectors to linearize the vectors, and the enzyme cutting reaction system is shown in Table 7; S2-4, recombination and linkage of the F2 fragments carrying the target gRNA to the linearized pENTR4+Null, pENTR4+tMet and pENTR4+tMetdDt vectors respectively; S2-5, After overnight culture, single clone was picked for PCR and sequencing, and after successful identification, the intermediate vectors pENTR4+Null, pENTR4+tMet and pENTR4+tMetdDt carrying double gRNA were obtained, the construction process is shown in Figure 2, and the vector transcription unit is shown in Figure 2; S3, Construction of mobile CRISPR / Cas9 gene editing vector; S3-1, The intermediate vector and the TLS carried by the target vector are one-to-one corresponding, and LR reaction is carried out, and the reaction system is shown in Table 8; S3-2, It is suggested to add the reaction system on ice, first add other components except LR Clonase II, mix thoroughly by vortexing for a short time twice, then vortex the LR Clonase II enzyme mixture for a short time twice (2 seconds each time), and take 2 μl and add to the reaction system; S3-3, Incubate at 25°C overnight, add 1 μl of proteinase K solution to each sample to terminate the reaction; S3-4, The reaction product is transferred into TOP10 competent cells and incubated overnight, and after successful PCR identification and sequencing, the mobile editing vector pMDC7+Cas9tMet+rbcS-e9t terminator+gRNAtMe and pMDC7+Cas9tMetdDt+rbcS-e9t terminator+gRNAtMetdDt of the target gene and the non-moving pMDC7+Cas9Null+rbcS-e9t terminator+gNIA1Null vector are obtained as a control group; The flow chart is shown in Figure 3; S3-5, Transfer all the above vectors into Agrobacterium rhizogenes K599, and after 48h culture, randomly pick single colonies on the plate, perform colony PCR identification, and after successful identification, preserve the bacteria for subsequent use.

2. Application of the mobile CRISPR / Cas9 gene editing fusion vector obtained according to 1 in combination with Agrobacterium rhizogenes-mediated plant hairy root transformation technology.

3. The positive seedlings obtained according to 2 are subjected to mobile editing vector mobility and editing type detection.

4. Application of the editing type detected according to 3 for gene function verification.

5. The mobile CRISPR / Cas9 gene editing fusion vector obtained according to 1 is used for gene function verification.

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