WO2022236939A1

WO2022236939A1 - Gene editing vector, method for editing gene by means of using same, and use thereof

Info

Publication number: WO2022236939A1
Application number: PCT/CN2021/103026
Authority: WO
Inventors: 许凯; 张望
Original assignee: 南京师范大学
Priority date: 2021-05-11
Filing date: 2021-06-29
Publication date: 2022-11-17
Also published as: CN113774081A; CN113774081B

Abstract

Disclosed are a gene editing vector, a method for editing a gene by means of using same, and the use thereof. The gene editing vector comprises a sequence from a 5'-UTR region to a capsid protein region of a virus of the Potyvirus genus, a gRNA, an insertion sequence and a 3'-UTR region, which are arranged in sequence; and the insertion sequence comprises a nucleic acid sequence encoding the viral capsid protein of the virus of the Potyvirus genus. By means of constructing a gene editing system by using the virus of the Potyvirus genus, various crops such as soybeans and potatoes that are less involved in the prior art can be gradually influenced, and the object and range of gene editing are expanded. Moreover, the technical problem of it being difficult to produce gRNA in a conventional way due to the fact that the virus of the Potyvirus genus cannot produce subgenomic RNA is further solved. Therefore, the gene editing vector has great significance in the technical field of genetic engineering.

Description

A gene editing vector and method and application thereof for editing genes

cross reference

This application claims the priority of the Chinese patent application No. 202110510966X filed on May 11, 2021 with the patent title "A Gene Editing Carrier and Its Method and Application for Editing Genes", the entire disclosure content of which is incorporated herein by reference in its entirety .

technical field

The invention relates to the technical field of genetic engineering, in particular to a gene editing vector and its gene editing method and application.

Background technique

In many bacteria and most archaea, there is a defense system that can be used to resist foreign DNA (such as phage), called CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Cas9 endonuclease) system (Bhaya et al. al., 2011). The CRISPR/Cas9 system consists of two basic components, one is the endonuclease Cas9 protein, and the other is gRNA (guide RNA). Under certain conditions, the gRNA can form a complex with the Cas9 protein, and through complementary base pairing, the gRNA can guide the Cas9 protein to the target DNA, and then the Cas9 protein can bind and cut the target double-stranded DNA, thereby causing the target DNA double-strand break ( double strand break). The DNA repair mechanism in the cell will repair the damaged double-stranded DNA, including NHEJ (non-homologous end-joining) and HDR (homology-directed repair) (Hsu et al., 2014). In most cases, NHEJ plays a major role, but the repair of double-stranded DNA mediated by the NHEJ repair mechanism is often imprecise, and may introduce different types of mutations in the repaired DNA double-strand, such as base insertion, Base deletion and base substitution, etc., may cause changes or deletions in the function of the target DNA, thereby achieving the purpose of "gene editing". Using the CRISPR/Cas9 system, people have realized the editing of the genomes of various organisms including animals, plants, and microorganisms, thereby achieving directional transformation of certain traits of target organisms. For example, a transient expression vector containing CRISPR/Cas9-related components is introduced into maize embryos by a gene gun, and the thermosensitive genic male-sterile 5 (TMS5) gene of maize is edited, so that at 32°C, male sterility can be obtained. characteristics of maize (Li et al., 2017). However, this type of gene editing strategy using the CRISPR/Cas9 system is based on transient expression vectors, and requires special external equipment (such as a gene gun) to introduce plasmids capable of expressing CRISPR/Cas9 components into the target. tissues or cells. Based on such methods, the number of cells that can obtain CRISPR/Cas9 components is very limited, and the amount of CRISPR/Cas9 components that enter the target cells is also limited. In order to obtain the target mutant, it often requires a lot of time and manpower to carry out related operations, such as obtaining plant immature embryos and performing tissue culture.

In recent years, in order to facilitate the introduction of CRISPR/Cas9 components into target tissues and cells, simplify the operation process, and improve the efficiency of gene editing, there have been reports on the use of plant virus vectors to introduce gRNA into target tissues and express them in large quantities, thereby achieving the purpose of plant gene editing. , such as using geminivirus, tobacco rattle virus (Tobacco rattle virus, TRV), tobacco mosaic virus (Tobacco mosaic virus, TMV) and barley stripe mosaic virus (Barley stripe mosaic virus, BSMV) to edit the host plant genome.

Taking the geminivirus wheat dwarf virus (WDV) as an example, WDV is a plant DNA virus. The authors used the modified WDV to express a large number of Cas9 proteins and DNA templates for HDR repair to achieve the endogenous genes. Gene targeting. Thanks to the efficient replication of the engineered WDV replicon in the target cells, its editing efficiency on target genes is 12 times that of non-viral vectors (Gil-Humanes et al., 2017). Both the motor protein and the capsid protein of the modified WDV virus are removed, thereby losing the ability to move, but able to replicate efficiently. Since WDV is a DNA virus, the authors used the WDV replicon to express a large amount of donor DNA and serve as a template for homologous recombination repair to achieve gene targeting of plant endogenous genes. The gene editing vector based on the WDV replicon can express Cas9 and gRNA in large quantities at the same time, and can edit the endogenous genes of corn, wheat, rice and other crops.

However, plant geminiviruses are DNA viruses. When infecting plants, geminiviruses may compete with host cells for replication, translation and other related factors, interfering with the normal growth of plants, and also bring difficulties to plant tissue regeneration. Gene editing vectors based on geminivirus replicons are often unable to mobilize and disperse in plants. In addition, geminiviruses are still one of the most serious threats to crops. They can cause severe symptoms and cause huge damage to crops, and geminiviruses can be transmitted by insects (such as whitefly). Once spread, it is difficult to control.

Tobacco rattle virus (TRV) is a positive-sense single-stranded RNA virus whose genome contains two RNA strands. By modifying TRV RNA2, the author successfully used TRV to express gRNA in Nicotiana benthamiana and cooperate with Cas9 protein to realize the editing of endogenous genes in Nicotiana benthamiana (Ali et al., 2015). The author destroyed the 2b protein (related to nematode transmission) and 2c protein (unknown function) expression boxes on TRV RNA2, and this modification did not affect the systematic movement of the virus on Nicotiana benthamiana. By cloning the gRNA to the downstream of the CP protein expression box on TRV RNA2, the transcription is driven by the pea early browning virus (PEBV) promoter (192 bp) of the same genus. Applying this strategy, it successfully edited the PDS and PCNA genes of the injected and systemic leaves of Nicotiana benthamiana, and the editing efficiency was about 50%.

However, the genome of TRV contains two single-stranded RNAs. Although the gene editing vector based on TRV maintains the ability to move systematically, the host range of TRV is relatively limited, and it generally does not infect important crops including barley, wheat, and corn. monocots, and cannot infect dicotyledonous crops such as soybean, thus limiting its application in these important crops.

In 2017, some researchers reported using Tobacco mosaic virus (TMV) to edit endogenous genes in Nicotiana benthamiana (Cody et al., 2017). TMV is also a single-stranded RNA virus. The author deleted the CP protein expression box of TMV. The TMV mutant lost the ability to move systematically in Nicotiana benthamiana, but still retained the ability to replicate. The method of Agrobacterium infiltration can Replication was performed on agroinfiltrated leaves of N. benthamiana. The authors cloned the gRNA behind the MP protein expression cassette and transcribed the gRNA from the subgenomic promoter of the CP. There is still about 60 nt redundant bases between the subgenomic promoter transcription start site of CP and the gRNA, and there is a UTR sequence of nearly 200 bp after the gRNA. The endogenous gene AGO1 of N. nicotianae was edited, and the efficiency can reach 70%. In addition, the author found that multiple gRNAs can be directly connected in series, and they can all play a role.

However, when it is designed, the motor protein of the virus itself is removed, so that the virus loses the ability to move systematically, so that the parts where it can play a role are relatively limited. In addition, TMV cannot infect wheat, corn and other gramineous crops, nor can it be transmitted through seeds, that is, it does not have the potential to directly edit T0 generation seeds and quickly harvest target mutants.

Barley Stripe Mosaic Virus (BSMV) belongs to the Plant Baculoviridae family and the genus Hordevirus. Its genome contains three positive-sense single-stranded RNAs, called RNAα, RNAβ and RNAγ. It is known that there are multiple sites on the genome of barley stripe mosaic virus that can insert foreign fragments without affecting the replication and movement of the virus itself, such as the 5' end and 3' end of γb; the 3' end of TGB3; in addition, the author found that The middle part of the CP sequence of barley stripe mosaic virus can be replaced by a foreign fragment without affecting the movement of the virus (Hu, Jiacheng, Li, et al., 2019, Molecular Plant Pathology), these positions can be used to insert and express gRNA . One of the sites preferred by the author, the 3' end of γb, is used for inserting and expressing gRNA. The gRNA comprises two parts, the spacer part at the 5' end and the backbone part at the 3' end next to each other; the nucleotide sequence of the spacer can be changed, and its length can also change according to the difference of the Cas9 protein. The upstream of the Spacer and/or the downstream of the backbone part can contain additional sequences in addition to the sequence on the genome of the barley stripe mosaic virus, which can be another or multiple gRNA sequences and/or other than the virus itself and the gRNA other sequences. BSMV-based gene editing vectors can express gRNA in large quantities and successfully edit endogenous genes in Nicotiana benthamiana, wheat and maize.

In 2020, a gene editing system using the plant negative-sense RNA virus Sonchus yellow net rhabdovirus (SYNV) was reported (Xiaonan Ma, et al., 2020, Nature Plants), in this system The gRNA is placed in the middle of the N/P gene in the positive strand RNA of the virus and the original sequence in the middle of the N/P gene is added to control its transcription, and the 5' and 3' ends of the gRNA are added with pre - The tRNA sequence helps the gRNA to be spliced out of the mRNA transcribed by the virus. In this system, a gene expressing Cas9 protein is added downstream of the gRNA, so that the virus can not only express the gRNA, but also express the Cas9 protein, realizing gene editing that does not depend on DNA but only on RNA (virus). However, due to the limited host range of SYNV, this system has only been proven effective in Nicotiana benthamiana.

In 2020, it was reported that the modified Tobacco rattle virus (TRV) gene editing system can enter seeds for editing (heritable) (Evan E. Ellison, et al., 2020, Nature Plants). This system is based on the TRV system reported in 2015, which not only uses the TRV RNA2 to remove the 2b and 2c expression cassettes, but also adds the same genus pea early browning virus (pea early browning virus, PEBV) promoter subgenomic RNA promoter (192bp) Drives transcription of the gRNA. However, unlike the report in 2015, this vector added the complete gene of the flowering gene Flowering LocusT (FT), or the gene mFT with a mutation in the start codon, or a truncated mutant downstream of the gRNA. Since fragments of the flowering gene can move systematically towards the growing point in the plant, it facilitates the delivery of the gRNA to the seed and increases the editing efficiency in the seed. However, the host range of TRV is relatively limited, and it cannot infect important crops such as barley, wheat, and corn.

In summary, most of the current gene editing systems that use plant viruses to deliver guide RNA rely on Cas9 transgenic plants (except SYNV), and the range of hosts is still not wide enough, and the hosts that can be applied are still limited.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a gene editing vector, a gene editing method and its application, specifically constructing a gene editing system through a virus of the genus Potatovirus with a very wide range of hosts and the same genome structure, which broadens the range of available genes. Edited object and scope.

In a first aspect, the present invention provides a gene editing vector, comprising: a sequence of potyviruses arranged in sequence from the 5'-UTR region to the capsid protein region, gRNA, insert sequence and 3'-UTR region;

The insertion sequence is optionally any of the following:

i) a nucleic acid sequence encoding the viral capsid protein of said potyvirus;

ii) After the nucleic acid sequence shown in i) is substituted, deleted and/or increased by one or more nucleotides, it can still form the nucleic acid sequence of the complete RNA element required for viral replication with the nucleic acid sequence in the 3'-UTR region.

The present invention adds gRNA, insertion sequence and 3'-UTR region after the stop codon of poty virus capsid protein. The insert sequence and the 3'-UTR region together form the complete RNA element (or promoter sequence) required for viral replication, enabling the virus carrying the gRNA to replicate normally and systematically infect the host plant, thereby infecting the infected tissue. gene editing.

Further, the sequence of the capsid protein region is codon-optimized to increase the free energy of the secondary structure of the nucleic acid sequence;

Preferably, compared with the sequence before optimization, the optimized sequence has no continuous completely identical homologous fragments longer than 30 bases.

The inventors optimized the codon for the nucleic acid sequence encoding the viral capsid protein in Potavirus genus virus, aiming at avoiding the same sequence of the newly inserted viral capsid protein after the stop codon to produce virus-mediated homologous recombination, thereby Let the gRNA between the two sequences be lost. However, judging from the conventional knowledge in the field, unoptimized viral capsid protein may also have a certain effect.

Further, the insertion sequence is processed through the following process i) or process ii):

i) selecting the nucleic acid sequence encoding the viral capsid protein of the potyvirus as an insertion sequence;

ii) Dividing the nucleic acid sequence encoding the viral capsid protein of the potyvirus into multiple sections of 100-300 nt, removing at least one nucleic acid sequence at random, and carrying out the remaining nucleic acid sequence in the vector Evaluation of viral replication ability and/or gene editing efficiency; if the replication and gene editing of the potyvirus can be carried out smoothly, the remaining nucleic acid sequence is the insertion sequence.

Specifically, based on the analysis of the secondary structure of the viral capsid protein CP and the sequence of the 3'-UTR, the inventors use the distribution characteristics of the neck ring structure on the secondary structure to avoid destroying the neck ring structure. , the complete nucleic acid sequence encoding the viral capsid protein is truncated step by step from the 5' end (each truncation is basically in the unit of 100-300nt), and the evaluation and screening are carried out according to the virus replication ability and/or gene editing efficiency; if The replication and gene editing of the potyvirus can be carried out smoothly, and the nucleic acid sequence left is the insertion sequence. In addition, the inventors found that some sequences truncated by this rule have higher editing efficiency than the complete viral capsid protein sequence.

Further, a nucleic acid sequence capable of self-cleavage or intracellular endonuclease cleavage is also included between the sequence of the capsid protein region and the gRNA, preferably a nucleic acid sequence encoding a ribozyme or a Pre-tRNA.

Further, the ribozyme is a mutant stigma ribozyme; preferably, the nucleotide sequence of the mutant stigma ribozyme includes the nucleotide sequence shown in SEQ ID NO.2.

The ribozyme preferably inserted here in the present invention is a mutant stigma-shaped ribozyme (Hammer head ribozyme), and other similar ribozymes in the prior art can also be used to release gRNA.

Further, the virus of the genus Potatovirus includes but is not limited to Potato virus Y (Potato virus Y), soybean mosaic virus (Soybean mosaic virus), common bean mosaic virus (Bean common mosaic virus), bean yellow mosaic virus ( Bean yellow mosaic virus), Blackeye cowpea mosaic virus, Cowpea aphid-borne mosaic virus, Cowpea green vein banding virus, Guar Guar symptomless virus, Mungbean mosaic virus, Pea seedborne mosaic virus, Peanut mottle virus, Peanut stripe virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, Watermelon mosaic virus, Sugarcane mosaic virus, Wheat streak mosaic virus mosaic virus), Turnip mosaic virus, Plum pox virus, or Sunflower mosaic virus.

Viruses of the potyvirus genus have the same genome structure (as shown in A in FIG. 3 ), and are theoretically applicable to the design idea of the gene editing vector provided by the present invention.

The design idea of the gene editing vector involved in the present invention can be applied to all viruses of the potyvirus genus.

Further, the gene editing vector includes the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

In a second aspect, the present invention provides a gene editing method, using the gene editing vector to perform gene editing on a target plant.

Further, the gene editing vector is transformed into the target plant by Agrobacterium infiltration, particle gun bombardment or friction inoculation.

Further, the target plant is a host plant that potyvirus can infect, including:

One or more of soybean, kidney bean, cowpea, mung bean, tobacco, watermelon, potato, alfalfa, sweet potato, corn, wheat or barley.

The present invention further provides the gene editing vector, or the application of the gene editing method in improving plant phenotype; the plant phenotype preferably includes: yield, disease resistance, stress resistance, seed quality and fruit quality one or more.

The gene editing vector provided by the present invention can be introduced into plant tissue cells by methods such as gene gun bombardment, Agrobacterium infiltration and friction inoculation, and can replicate and move in Nicotiana benthamiana; the gene editing vector contains gRNA sequences, Under the condition of transient or transgenic expression of Cas protein, genome editing of plants such as Nicotiana benthamiana can be realized. For example, the gene editing vector based on soybean mosaic virus is inoculated on Nicotiana benthamiana with the method of Agrobacterium infiltration, and under the condition of the presence of spCas9 protein, it can directly inoculate the virus system in the area. Infected plant tissue for gene editing.

The present invention has following beneficial effect:

The present invention constructs a gene editing system based on Potavirus genus virus. Compared with the existing gene editing vector based on TRV, the gene editing vector provided by the present invention can infect some important crops, including soybean, potato and alfalfa Wait;

Compared with the TMV-based gene editing vector, the gene editing vector provided by the present invention retains the systemic motility and replication ability of the virus in the host plant, so that gene editing of a wider range of plant tissue cells can be achieved, not just limited In the field of virus inoculation on plants;

Compared with gene editing vectors based on geminiviruses, the gene editing vectors provided by the present invention will not compete with plant cells for elements such as DNA replication and interfere with the normal plant cell cycle under the premise of achieving higher editing efficiency; in addition, potato Viruses of the genus Y virus, as RNA viruses, will not integrate into the plant genome and introduce additional foreign fragments into the plant genome;

Compared with BSMV-based gene editing vectors, Potavirus genus has a wide host range and has the potential to edit a wider range of host plants, including soybean (soybean), kidney bean (bean), cowpea (cowpea), mung bean (mungbean) , watermelon (watermelon), alfalfa (alfalfa), sweet potato (sugarcane) and so on.

The gene editing vector provided by the present invention can insert the ribozyme sequence, gRNA sequence and the complete or partial nucleic acid sequence encoding the viral capsid protein into the stop codon of the nucleic acid sequence of the viral capsid protein without affecting the potyvirus genus virus in Systemic infection on the host plant Nicotiana benthamiana; additional nucleotide sequences can be added to both ends of the gRNA sequence without disrupting its normal function; at the same time, since the virus can move in the plant, this gene editing vector can outside plant tissue for editing.

The present invention provides a gene editing vector under the condition of transient or transgenic expression of Cas9, which can be used for transient expression of Cas9 on the inoculated leaves of Nicotiana benthamiana through PCR/RE experiments on the sixth day after inoculation The mGFP5 gene of the 16c transgenic N. benthamiana or the PDS gene of the Cas9 transgenic N. benthamiana achieved an editing efficiency of more than 50%. On the 12th day of inoculation, the PCR/RE experiment was performed and calculated using the quantity one software. The mGFP5 gene of the 16c transgenic N. benthamiana transiently expressing Cas9 or the PDS gene of the Cas9 transgenic N. benthamiana can achieve an editing efficiency of more than 50% on the leaves.

Description of drawings

Fig. 1 is a schematic diagram of the structure of a soybean mosaic virus-based gene editing vector provided in Example 1 of the present invention.

Fig. 2 is a schematic diagram of the RNA secondary structure analysis of soybean mosaic virus original capsid protein sequence plus 3'UTR sequence using mfold provided by Example 1 of the present invention; where the arrows indicate the truncation positions.

Figure 3 is a schematic diagram of the genome structure comparison of the potyvirus genus virus before and after transformation provided in Example 1 of the present invention; wherein A is a schematic diagram of the genome structure of the potyvirus genus virus; B is the transformed potyvirus genus virus genome for gene editing Schematic diagram of the structure; C is a schematic diagram of the gene structure of the soybean mosaic virus used for gene editing and the primers required for the construction of this clone; D is a schematic diagram of the gene structure of the potato virus Y used for gene editing and the primers required for the construction of the clone; among them, CP* It is the codon-optimized sequence of the capsid protein region of Potavirus genus virus, Rz refers to ribozyme or other nucleic acid sequences with self-cleaving function, target sequence and spCas9-gRNA scaffold refer to the target sequence that constitutes the gRNA and backbone sequence, CP sequence refers to the insert sequence (the nucleic acid sequence of the full-length or truncated viral capsid protein), original 3'-UTR refers to the 3'UTR sequence of the poty virus before optimization; Rz, gRNA, insert The sequence together with the original 3'-UTR constitutes the 3'UTR sequence (New 3'-UTR) of the transformed virus.

Fig. 4 is a schematic diagram of the Western blotting results provided in Example 1 of the present invention to detect the accumulation of soybean mosaic virus inserted into gRNA in different versions and wild-type soybean mosaic virus on the 6th day of injection of leaf CP.

Figure 5 is a schematic diagram of the results of agarose gel electrophoresis detection of different versions of gRNA-inserted soybean mosaic virus injection gene editing efficiency on the 6th day provided by Example 1 of the present invention; wherein, NdeI of the mGFP5 gene fragment was destroyed due to gene editing Restriction site, so the edited gene fragment cannot be digested by NdeI.

Figure 6 is the sequencing result of the mGFP5 editing site in the leaf cells of the injection area on the 6th day of the version 7 (SMV-HHRz-gRNA-insert sequence #3) using ribozyme provided by Example 1 of the present invention; wherein CATATG is the NdeI restriction site , CGG is PAM; the horizontal bar represents the deletion mutation, and the bold represents the insertion mutation.

Figure 7 shows the sequencing results of the mGFP5 editing site in the leaf cells of the injection area on the 6th day of the version 8 (SMV-HHRz-gRNA-insert sequence #4) using ribozyme provided by Example 1 of the present invention; wherein CATATG is the NdeI restriction site , CGG is PAM; the horizontal bar represents the deletion mutation, and the bold represents the insertion mutation.

Figure 8 is a schematic diagram of the results of Western blotting detection of virus CP accumulation in leaves of different versions of soybean mosaic virus inserted with gRNA and wild-type soybean mosaic virus system on day 12 provided by Example 1 of the present invention.

Fig. 9 is the version 7 (SMV-HHRz-gRNA-insert sequence #3) and version 8 (SMV-HHRz-gRNA-insert sequence #3) and version 8 (SMV-HHRz-gRNA-insert sequence) of soybean mosaic virus nuclease detected by agarose gel electrophoresis provided in Example 1 of the present invention #4) Schematic diagram of gene editing efficiency results in systemically infected leaf cells on day 12; where, because the gene editing destroyed the NdeI restriction site of the mGFP5 gene fragment, the edited gene fragment could not be digested by NdeI .

Figure 10 is the sequencing result of the mGFP5 editing site in the leaf cells of the soybean mosaic virus version 7 (SMV-HHRz-gRNA-insert sequence #3) provided in Example 1 of the present invention on the 12th day; wherein CATATG is NdeI enzyme The cleavage site, CGG is PAM, and the horizontal bar represents the deletion mutation.

Figure 11 is the sequencing result of the mGFP5 editing site in the leaf cells of the soybean mosaic virus version 8 (SMV-HHRz-gRNA-insert sequence #4) provided in Example 1 of the present invention on the 12th day; wherein CATATG is NdeI enzyme The cleavage site, CGG is PAM, and the horizontal bar represents the deletion mutation.

Fig. 12 is a schematic diagram of the structure of the gene editing vector based on potato virus Y provided by Example 2 of the present invention.

Figure 13 is a schematic diagram of the results of western blotting detection of gRNA-inserted Potato virus Y and wild-type Potato virus Y injection leaf CP accumulation on day 6 provided by Example 2 of the present invention; wherein, NcoI of the PDS gene fragment was destroyed due to gene editing Restriction site, so the edited gene fragment cannot be digested by NcoI.

Fig. 14 is a schematic diagram of the results of agarose gel electrophoresis detection of gene editing efficiency of gRNA-inserted potato virus Y injection leaves on day 6 provided in Example 2 of the present invention.

Figure 15 is the sequencing result of the PDS editing site of the injection leaf on the 6th day provided by Example 2 of the present invention; wherein, CCATGG is the NcoI restriction site, GGG is PAM, the horizontal bar represents the deletion mutation; the bold represents the insertion mutation, and the italic represents point mutation.

Fig. 16 is a schematic diagram of the results of Western blotting detection of the accumulation of leaf CP of the gRNA-inserted Potato virus Y and wild-type Potato virus Y system on the 12th day provided by Example 2 of the present invention.

Figure 17 is a schematic diagram of the results of agarose gel electrophoresis detection of the gene editing efficiency of the potato virus Y system inserted into the gRNA on the 12th day provided by Example 2 of the present invention; wherein, the NcoI restriction site of the PDS gene fragment was destroyed due to gene editing , so the edited gene fragment cannot be digested by NcoI.

Figure 18 is the sequencing result of the PDS editing site in the 12th day system leaf provided by Example 2 of the present invention; wherein CCATGG is the NcoI restriction site, GGG is PAM; the horizontal bar represents the deletion mutation, the bold represents the insertion mutation, and the italic represents the point mutation.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

This example constructs a CRISPR/Cas9 gene editing system using soybean mosaic virus as a carrier. The system is shown in Figure 1, and the construction process is as follows:

1. Choose soybean mosaic virus SC7 isolate (Genbank Accession#: MH919385) as raw material;

2. Codon-optimized nucleic acid sequence encoding capsid protein of soybean mosaic virus SC7 isolate. Using the online website (https://www.vectorbuilder.cn/tool/codon-optimization.html), one-click optimization of species selection common tobacco, the optimized nucleic acid sequence is obtained, as shown in SEQ ID NO.1, the optimized The nucleic acid sequence of soybean mosaic virus capsid protein was directly synthesized to replace the original CP sequence in the reading frame. (Note: The role of codon optimization is to avoid coincidence with the newly inserted CP sequence after the stop codon, resulting in virus-mediated homologous recombination, so that the gRNA in the middle of the two sequences is lost. But we still speculate that the non-optimized CP Might also work to some extent.)

3. The soybean mosaic virus is divided into 6 segments for PCR amplification, and the mutant hammerhead ribozyme, gRNA (edited mGFP5, sequence shown in SEQ ID NO.48), and the insert sequence are integrated into soybean by PCR Among the infectious clones of mosaic virus, there are a total of 8 different versions. Wherein, insert sequence is its secondary structure analysis (Fig. 2) according to the sequence of viral capsid protein CP and 3'-UTR together, through the distribution characteristic of the neck ring structure on its secondary structure, in order not to destroy main Based on the principle of the independent neck loop structure, the complete nucleic acid sequence encoding the viral capsid protein is truncated from the 5' end (in units of 100-300nt), and the truncated position is shown by the arrow in Figure 2.

The overall concept of the eight different versions is shown in Figure 3 C. Specifically, the construction methods of the different versions are as follows:

(1) Versions 1-4 do not contain hammerhead ribozymes, only gRNA and insert #1-4;

(2) Versions 5-8 contain hammerhead ribozymes, along with gRNA and insert #1-4.

Versions

1 and 5 contain Insert #1,

Versions

2 and 6 contain Insert #2,

Versions

3 and 7 contain Insert #3, and

Versions

4 and 8 contain Insert #4. Wherein, insertion sequence #1, insertion sequence #2 and insertion sequence #3 are truncated soybean mosaic virus capsid protein nucleic acid sequences, and insertion sequence #4 is a full-length soybean mosaic virus capsid protein nucleic acid sequence.

The inserted hammerhead ribozyme sequence is shown in SEQ ID NO.2. The nucleic acid sequence of the inserted sequence #1-4 is shown in SEQ ID NO.44-47, and the inserted gRNA backbone sequence is shown in SEQ ID NO.48.

The specific cloning steps are as follows:

(1) Using the complementary DNA (cDNA) of the soybean mosaic virus SC7 isolate as a template, fragment 1 was amplified by primers SMV/PVY-frag1-F and SMV-frag1-R, and by primers SMV-frag2-F and SMV- fragment 2 was amplified by frag2-R, fragment 3 was amplified by primers SMV-frag3-F and SMV-frag3-R, an intermediate product was amplified by primers SMV-frag4-F and SMV-frag4-R1, and the intermediate product was recovered by tapping As a template, fragment 4 was amplified by primers SMV-frag4-F and SMV-frag4-R2.

(2) Using the optimized soybean mosaic virus capsid protein nucleic acid sequence after gene synthesis as a template, the fragment is amplified by primers SMV-frag5-F and SMV-frag5a-R1, SMV-frag5a-R2, SMV-frag5a-R3 5a; Fragment 6a was amplified by primers SMV-frag6a-F and SMV/PVY-frag6-R using the infective DNA clone of soybean mosaic virus as a template, and fragment 6a was amplified by primers SMV-frag6b-F and SMV/PVY-frag6-R Fragment 6b was amplified, fragment 6c was amplified by primers SMV-frag6c-F and SMV/PVY-frag6-R, and fragment 6d was amplified by primers SMV-frag6d-F and SMV/PVY-frag6-R.

(3) Fragment 5a is mixed with fragments 6a, 6b, 6c or 6d as templates, respectively amplified by primers SMV-frag5-F and SMV/PVY-frag6-R to obtain fragments 5a+6a, 5a+6b, 5a+6c , 5a+6d. Mix pCB301-314 and

fragments

1, 2, 3, 4 after StuI and SmaI double digestion with fragments 5a+6a, 5a+6b, 5a+6c, 5a+6d respectively, and obtain clone version 1 by yeast homologous recombination to 4 (see C in Figure 3 for a schematic diagram of cloning construction).

(4) Using the optimized soybean mosaic virus capsid protein nucleic acid sequence of gene synthesis as a template, through primers SMV-frag5-F and SMV-frag5b-R1, SMV-frag5b-R2, SMV-frag5b-R3, SMV- frag5a-R2, SMV-frag5a-R3 amplified fragment 5b.

(5) Fragment 5b was mixed with fragments 6a, 6b, 6c, and 6d as templates, and fragments 5b+6a, 5b+6b, 5b+6c were amplified by primers SMV-frag5-F and SMV/PVY-frag6-R respectively , 5b+6d.

(6) Mix pCB301-314 and

fragments

1, 2, 3, and 4 after double digestion with StuI and SmaI with fragments 5b+6a, 5b+6b, 5b+6c, and 5b+6d, respectively, and obtain them by yeast homologous recombination Clone versions 5 to 8 (see C in Figure 3 for a schematic diagram of the clone construction).

4. Extract a large number of yeast plasmids of cloned version 1-8, the specific method is as follows: select a single colony that grows and place it in 50ml SC-T-, shake the bacteria overnight at 30°C and 250rpm. Centrifuge at 5000rpm for 5min, remove the supernatant, add 1.5ml sorbitol phosphate buffer solution (0.1M PBS containing 1.2M sorbitol, pH=7.4) for every 1g of yeast pellet, 100ul helicase solution (0.12g/ml, helicase powder Dissolve in a mixed liquid containing 10% 0.1M PBS, 40% sterile water, and 50% glycerol), 10μl RNaseA (10mg/ml), vortex and mix well, and enzymatically digest at 37°C overnight. After the yeast cell wall is removed, the plasmid can be extracted by conventional alkaline lysis method, and a plasmid extraction kit (Novazyme, Nanjing, China) is used in this implementation case.

5. Prepare Agrobacterium strain C58C1 competent with _CaCl2 treatment method, and then use liquid nitrogen freeze-thaw method to transform yeast plasmid into newly prepared competent C58C1, the specific method is as follows:

The activated C58C1 was shaken in 2ml LB (containing 50μg/ml streptomycin and 100μg/ml rifampicin) at 30°C and 250rpm overnight. Add this 2ml bacterial solution into 50ml LB (without antibiotics), shake the bacteria at 30°C and 250rpm until OD=0.5. Put the bacterial solution on ice for 10 min, then 5000 rpm, 4°C, 10 min, remove the supernatant, and resuspend the pellet in 1 ml of pre-cooled 20 mM CaCl ₂ . Add 100 μl of competent cells to the centrifuge tube previously added with yeast plasmid, place on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, place at 37°C for 5 minutes, place on ice for 5 minutes, add 1ml LB, and place at 30°C for 3 hours. 15000rpm, 30s, leave 100μL of supernatant to mix and precipitate, spread on solid LB medium containing antibiotics, put at 30°C for 4d, a single colony grows.

Pick a single colony and shake the bacteria overnight in LB medium, centrifuge and remove the supernatant to collect the bacteria, suspend the bacteria in MMA, inject the transgenic N. benthamiana expressing mGFP5, and inject Cas9-transformed Cas9 in the injection area on the 3rd day after injection The pKSE401-Cas9 vector of Agrobacterium, 6 days after injection, the injected leaves were taken by Western blotting to detect the accumulation of SMV CP (Figure 4), and it was found that all versions can replicate, and the

versions

3 and 7 containing the insertion sequence #3 have the strongest replication ability , can achieve the same level of replication as wild-type virus.

Take the injected leaves to extract DNA, use primers mGFP5/F and mGFP5/R PCR to amplify mGFP5, and then digest the product with restriction endonuclease NdeI overnight. Gel electrophoresis detection of gene editing efficiency (Figure 5) found that version 7 (SMV-HHRz-gRNA-insert sequence #3) containing hammerhead ribozyme and insert sequence #3 had the highest gene editing efficiency, containing hammerhead ribozyme and insert sequence #3 The version 8 of insert sequence #4 (SMV-HHRz-gRNA-insert sequence #4) was next; the product after digestion was gel-tapped and purified, then connected to the T vector for sequencing, and the sequencing results were compared with the original gene sequence (Figure 6, Figure 7), verifying that mGFP5 in injected leaves was indeed gene edited.

On the 9th day after injection, the upper leaves were injected with Agrobacterium transformed with the pKSE401-Cas9 vector expressing Cas9, and after another 3 days (12 days after injection), the upper leaves injected with Cas9 were taken for Western blotting to detect the accumulation of SMV CP ( Figure 8), all versions 1-8 were found to be able to move systematically, with the highest virus accumulation in version 7 (SMV-HHRz-gRNA-insert #3) containing the hammerhead ribozyme and insert #3, and version 8 (SMV-HHRz-gRNA-insert #4) followed.

Extract the DNA of upper leaves of

clones

7 and 8, PCR amplify mGFP5, and digest the product with restriction endonuclease NdeI overnight. The digested product is detected by agarose gel electrophoresis after phenolform extraction and ethanol precipitation Gene editing efficiency (Figure 9); the product after this enzyme digestion was tapped and purified, then connected to the T vector for sequencing, and the sequencing results were compared with the original gene sequence (Figure 10, Figure 11), and the systems of

versions

7 and 8 were verified to be mGFP5 are indeed gene edited.

At the same time, the present invention carried out an experiment process similar to that of Example 1 for soybeans, and obtained similar results, and successfully realized the gene editing of soybeans.

The primers used in this example are shown in the table below:

The primer sequences used in Table 1 Example 1

Example 2

In this example, a CRISPR/Cas9 gene editing system using potato virus Y as a carrier is constructed. The system is shown in Figure 12, and the construction process is as follows:

1, choose potato virus Y ZT5 isolate (Genbank Accession#: MF960848) as raw material;

2. Codon optimization of the nucleic acid sequence encoding the capsid protein of the potato virus Y ZT5 isolate. Using the online website (https://www.vectorbuilder.cn/tool/codon-optimization.html), one-click optimization of species selection common tobacco, the optimized nucleic acid sequence (SEQ ID NO.49), optimized potato Y Direct gene synthesis of viral capsid protein nucleic acid sequence.

3. Divide Potato virus Y into 5 segments for PCR amplification, and at the same time, hammerhead ribozyme, gRNA (editing PDS), and insert sequences are integrated into the infectious clone of Potato virus Y by PCR. The specific cloning steps are as follows :

(1) Using the potato virus Y infectious DNA clone as a template, fragment 1 was amplified by primers SMV/PVY-frag1-F and PVY-frag1-R, and amplified by primers PVY-frag2-F and PVY-frag2-R Fragment 2, the intermediate product was amplified by primers PVY-frag3-F and PVY-frag3-R1, and the intermediate product was recovered from rubber tapping as a template, and fragment 3 was amplified by primers PVY-frag3-F and PVY-frag3-R2;

(2) Using the optimized potato virus Y capsid protein nucleic acid sequence of gene synthesis as a template, through primers PVY-frag4-F and PVY-frag4-R1, PVY-frag4-R2, PVY-frag4-R3, PVY-frag4 -R4, PVY-frag4-R5, PVY-frag4-R6 amplified fragment 4; Potato virus Y infective DNA clone was used as a template to amplify fragment 5 by primers PVY-frag5-F and SMV/PVY-frag6-R ;

(3) Fragment 4 and fragment 5 were mixed as a template, and fragment 4+5 was amplified by primers PVY-frag4-F and SMV/PVY-frag6-R.

(4) Mix pCB301-314 and

fragments

1, 2, 3, 4+5 after double digestion with StuI and SmaI, and obtain a potato virus Y invasive DNA clone with gRNA inserted through yeast homologous recombination (see D) in Figure 3.

4. The procedure for extracting the yeast plasmid and transforming Agrobacterium C58C1 is the same as constructing the CRISPR/Cas9 gene editing system (Example 1) using soybean mosaic virus as a vector.

5. After a single colony grows, pick a single colony and shake the bacteria overnight in LB medium. After centrifugation, remove the supernatant to collect the bacteria. The bacteria are suspended in MMA and injected with transgenic spCas9-expressing Nicotiana benthamiana. Take the injection on the 6th day after injection. Leaf Western blotting detected the accumulation of PVY CP (Figure 13), PVY inserted with hammerhead ribozyme and gRNA can reach the same replication level as wild-type PVY.

Take the injected leaves to extract DNA, amplify the PDS with primers PDS/F and PDS/R PCR, and the product is digested overnight with the restriction endonuclease NcoI. Gel electrophoresis was used to detect gene editing efficiency (Figure 14). The results showed that some PDS gene fragments amplified from PVY-HHRz-gRNA-complete CP-infected plant tissues could not be enzymatically digested, suggesting that this part of the PDS gene was edited; The product after enzyme digestion was tapped and purified, then connected to the T carrier for sequencing, and the sequencing results were compared with the original gene sequence (Figure 15), to verify that the injected leaf PDS was indeed gene-edited;

On the 12th day after injection, the leaves of the system were taken to detect the accumulation of PVY CP by Western blotting (Figure 16); the DNA of the leaves of the system was extracted, and the product after PCR amplification of PDS was digested overnight with the restriction endonuclease NcoI, and the product after digestion was passed through phenol After imitation extraction and ethanol precipitation, agarose gel electrophoresis was used to detect the gene editing efficiency (Figure 17); the product after enzyme digestion was tapped and purified, then connected to the T carrier for sequencing, and the sequencing results were compared with the original gene sequence (Figure 18 ), verifying that the systemic leaf PDS is indeed gene-edited. At the same time, the present invention carried out an experiment process similar to that of Example 2 for potatoes, and obtained similar results, successfully realizing the gene editing of potatoes.

The primer sequences used in this example are shown in the table below:

The primer sequence used in table 2 embodiment 2

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Industrial Applicability

The invention provides a gene editing vector and its gene editing method and application. The gene editing vector comprises the sequence of the potato virus genus virus from the 5'-UTR region to the capsid protein region, gRNA, insert sequence and 3'-UTR region arranged in sequence; the insert sequence includes: encoding the potato Y The nucleic acid sequence of the viral capsid protein of a virus of the genus Virus. The present invention constructs a gene editing system through the virus of the genus Potatovirus, which can infect various crops such as soybeans and potatoes, which are less involved in the prior art, and expands the objects and scope of gene editing. At the same time, it also solves the technical problem that it is difficult to produce gRNA by conventional methods due to the inability of viruses of the genus Potatovirus to produce subgenomic RNA, which is of great significance in the field of genetic engineering technology.

Claims

A gene editing vector, characterized in that it comprises: a sequence of potyviruses arranged in sequence from the 5'-UTR region to the capsid protein region, gRNA, insert sequence and 3'-UTR region;

The insertion sequence is optionally any of the following:

i) a nucleic acid sequence encoding the viral capsid protein of said potyvirus;

ii) After the nucleic acid sequence shown in i) is substituted, deleted and/or increased by one or more nucleotides, it can still form the nucleic acid sequence of the complete RNA element required for viral replication with the nucleic acid sequence in the 3'-UTR region.
The gene editing vector according to claim 1, wherein the sequence of the capsid protein region undergoes codon optimization to change its nucleic acid sequence;

Preferably, compared with the sequence before optimization, the optimized sequence has no continuous completely identical homologous fragments longer than 30 bases.
The gene editing vector according to claim 1 or 2, wherein when the insertion sequence is selected from ii), the insertion sequence is obtained through the following procedures:

The nucleic acid sequence encoding the viral capsid protein of the potyvirus is divided into multiple segments of 100-300 nt, at least one nucleic acid sequence sequence is randomly removed, and the remaining nucleic acid sequence is carried out in the vector for viral replication Evaluation of ability and/or gene editing efficiency; if the replication and gene editing of the potyvirus can be carried out smoothly, the remaining nucleic acid sequence is the insertion sequence.
According to the gene editing vector according to any one of claims 1-3, it is characterized in that, between the sequence of the capsid protein region and the gRNA, there is also a gene that can cut itself or be cut by an intracellular endonuclease. The nucleic acid sequence is preferably a nucleic acid sequence encoding a ribozyme or a Pre-tRNA.
The gene editing vector according to claim 4, wherein the ribozyme is a mutant stigma ribozyme; preferably, the nucleotide sequence of the mutant stigma ribozyme is as shown in SEQ ID NO.2 .
The gene editing vector according to claim 1, wherein the virus of the genus Potatovirus comprises potato virus Y (Potato virus Y), soybean mosaic virus (Soybean mosaic virus), common bean mosaic virus (Bean common mosaic virus), Bean yellow mosaic virus, Blackeye cowpea mosaic virus, Cowpea aphid-borne mosaic virus, Cowpea green vein band virus green vein banding virus), Guar symptomless virus, Mungbean mosaic virus, Pea seedborne mosaic virus, Peanut mottle virus, Peanut stripe virus, Maize dwarf mosaic virus, Alfalfa mosaic virus, Watermelon mosaic virus, Sugarcane mosaic virus One or more of , Wheat streak mosaic virus, Turnip mosaic virus, Plum pox virus, and Sunflower mosaic virus.
The gene editing vector according to claim 1, wherein the gene editing vector comprises the nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.4.
A gene editing method, characterized in that it comprises: using the gene editing vector according to any one of claims 1-7 to perform gene editing on a target plant;

The target plants include one or more of soybean, kidney bean, cowpea, mung bean, tobacco, watermelon, potato, alfalfa, sweet potato, corn, wheat or barley.
The gene editing method according to claim 8, comprising:

The gene editing vector is transformed into the target plant by Agrobacterium infiltration, gene gun bombardment or friction inoculation.
The gene editing vector described in any one of claims 1-7, or the application of the gene editing method described in claim 8 or 9 in improving plant phenotypes; said plant phenotypes include: yield, disease resistance, resistance One or more of inverseness, seed quality and fruit quality.