CN105132451B - A kind of single transcriptional units directed modification skeleton carrier of CRISPR/Cas9 and its application - Google Patents

Abstract

The invention belongs to gene engineering technology fields, and in particular to a kind of single transcriptional units directed modification skeleton carrier of CRISPR/Cas9 and its application.The technical problem to be solved by the present invention is existing CRISPR/Cas9 genome editor system species versatility is low and is difficult to realize the collaboration of Cas9 protein expression and gRNA transcription.The technical scheme is that the building single transcriptional units skeleton carrier of CRISPR/Cas9, the transcription of Cas9 and guide RNA core cell are regulated and controled by a promoter.The present invention also provides construct the method for the special sex modification Cas9-gRNA recombinant vector of target site using the single transcriptional units skeleton carrier of CRISPR/Cas9.The present invention provides a kind of efficient single transcriptional units skeleton carriers of CRISPR/Cas9, the collaboration transcription that can effectively realize Cas9 the and gRNA unit based on the driving of Pol II type promoter, carries out simple, quick, efficient genome for a variety of eucaryotes and orients genetic modification.

Description

A CRISPR/Cas9 single transcription unit directional modification backbone vector and its application

technical field

The invention belongs to the technical field of genetic engineering, in particular to a CRISPR/Cas9 single transcription unit directional modification backbone vector and its application.

Background technique

In recent years, with the advancement of model animal and plant genome sequencing projects and the improvement of related gene manipulation technologies, sequence-specific nucleases (zinc-finger nuclease, ZFN; transcriptionactivator-like effectors nuclease, TALEN; clustered Regularly interspaced shortpalindromic repeats/CRISPR-associated protein-9, CRISPR/Cas9), causing DSBs at specific sites in the target organism's genome, and under the action of the organism's endogenous DNA repair system, different types of targeted genetic modification of the target genome can be achieved.

In the CRISPR/Cas9 genome editing system, the specific cleavage of the genome target sequence by CRISPR/Cas9 mainly depends on the formation of the crRNA and Cas9 protein in the guide RNA (guided RNA, gRNA) (a single guide RNA single strand after the fusion of tracrRNA and crRNA). The ribonucleoprotein complex recognizes the PAM (protospacer adjacent motif) on the target sequence (5'-NGG-3' is the characteristic of PAM) and its adjacent 20bp specific target sequence (protospacer) . In the experimental operation, an expression vector containing Cas9 protein and gRNA unit needs to be constructed. Through various transformation schemes, Cas9 protein expression and gRNA unit transcription are carried out in the target biological cells, and then the ribonucleoprotein complex of Cas9 protein and gRNA is recognized, combined, Cut specific sites in the target organism's genome, and then achieve different types of targeted genetic modifications under the action of the cell's endogenous DNA repair pathway.

In the currently used CRISPR/Cas9 genome editing system, Cas9 and gRNA are constructed in independent transcription and expression units: Cas9 protein expression unit consists of Pol II type promoter, Cas9 ORF, and terminator in turn, and Cas9 ORF needs to be fused with NLS sequence The gRNA transcription unit is sequentially composed of Pol III type promoter (U6, U3 and other small RNA transcription promoters are mostly used), gRNA unit (mostly tracrRNA+crRNA fusion gRNA unit published by Hwang et al. 2013), and Poly T terminator. Cas9 protein expression and gRNA transcription units can be constructed separately on two independent vectors for co-transformation, or can be constructed as two independent units on a single vector for transformation. Among them: the expression of Cas9 protein can be based on the characteristics of the transformation target and experimental requirements to select the appropriate Pol II type promoter, such as CMV, hsP70, SV40 commonly used in animal cells and commonly used in plants CaMV35S, ZmUb1, AtUb10 and other promoters; gRNA The transcription unit generally uses the specific U6, U3 and other small RNA transcription promoters in the target genome according to the transformation target.

However, in the above CRISPR/Cas9 genome editing system, the design of the independent Cas9 protein expression unit and gRNA transcription unit has inherent defects, and it is difficult to achieve the synergy of Cas9 protein expression and gRNA transcription. At the same time, since the transcription of the gRNA unit basically depends on Pol III-type promoters such as U6 and U3 with strong species preference, it is necessary to screen specific Pol III-type small RNA transcription promoters for different target organisms, and it is also difficult to achieve spatiotemporal specificity and induction. Transcriptional regulation greatly limits the efficiency and application scope of CRISPR/Cas9 in genome-directed modification.

SUMMARY OF THE INVENTION

The technical problems to be solved by the present invention are: the existing CRISPR/Cas9 genome editing system is difficult to achieve the synergy of Cas9 protein expression and gRNA transcription; specific small RNA transcription promoters need to be screened for different target organisms; it is difficult to achieve spatiotemporal specificity and induce transcription regulation.

The technical scheme of the present invention is a CRISPR/Cas9 single transcription unit backbone vector, and a promoter is used to regulate the transcription of Cas9 and the core unit of guide RNA.

Specifically, the core units are Cas9 ORF-Poly A-RZ cleavagesite-gRNA cloning scaffold-RZ cleavage site-RZ in order from 5' to 3', and there is at least one gRNA cloning scaffold, and there are at least one gRNA cloning scaffold between gRNA cloning scaffolds. RZ cleavage site; among them, Cas9 ORF is the Cas9 protein coding frame, RZ cleavage site is the ribozyme recognition cleavage site, gRNA cloning scaffold (abbreviated as gRNA CS) is the gRNA cloning and transcription unit, and RZ is the ribozyme holoenzyme RiboZyme.

Specifically, the number of RNA clone and transcription units is 1-5.

Specifically, the Cas9 protein coding frame also contains the nuclear localization signal NLS sequence, which has the amino acid sequence shown in Seq ID No.1.

Specifically, the nucleotide sequence of the Cas9 protein coding frame is shown in Seq ID No.2.

Specifically, the gRNA cloning and transcription unit has the nucleotide sequence shown in Seq ID No.3.

Specifically, a BsaI-ccdB-BsaI unit was also fused to the 5' end of the gRNA cloning and transcription unit, and the fusion had the nucleotide sequence shown in Seq ID No.4.

Specifically, the ribozyme holoenzyme and the corresponding recognized ribozyme recognition cleavage site are used in combination in the following ways:

(a) Substitute, delete or add one or more nucleotides to the nucleotide sequence of the ribozyme recognition cleavage site, and still be recognized and cut by the ribozyme holoenzyme;

Or (b) replacing the ribozyme holoenzyme, and at the same time corresponding to the replacement ribozyme recognition cleavage site, the replaced ribozyme recognition cleavage site is recognized and cut by the replaced ribozyme holoenzyme.

Specifically, the ribozyme holoenzyme is hammerhead ribozyme, class I intron, RNaseP, hairpin ribozyme, hepatitis D ribozyme or VS ribozyme.

Specifically, the ribozyme holoenzyme is a hammerhead ribozyme, and its encoding nucleotide has the sequence shown in Seq ID No.6.

Specifically, the ribozyme recognition cleavage site is recognized and cleaved by a hammerhead ribozyme, (a) having the nucleotide sequence shown in Seq IDNo.5;

Or (b) one or several nucleotides are substituted, deleted or added in the nucleotide sequence of (a), and can still be recognized and cleaved by hammerhead ribozymes.

Specifically, the Cas9-poly A-RZ cleavage site-gRNA cloning scaffold-RZcleavage site-RZ has the nucleotide sequence shown in Seq ID No.7.

Specifically, the promoter is a Pol II type promoter.

Specifically, the Pol II type promoter is cauliflower mosaic virus 35S promoter CaMV35S, maize Ubi1 promoter ZmUbi1, Arabidopsis thaliana Ubi10 promoter AtUbi10, cytomegalovirus CMV, heat shock protein 70 promoter hsP70 or monkey vacuolar Virus 40 promoter SV40.

The present invention also provides a preparation method for the specific modification of the Cas9-gRNA recombinant vector for the target site, comprising the following steps:

a. Identify the target DNA region of a specific biological genome, analyze the region with PAM characteristics, and select the 15-30bp DNA sequence adjacent to the 5' end of the PAM structure as the specific target sequence;

b. According to the selected specific target sequence, a forward oligonucleotide chain with 5'-CGGA-N _X -3' characteristics and a reverse oligonucleotide with 5'-AAAC-N _X -3' characteristics are synthesized respectively. Nucleotide chain, N represents any one of A, G, C, T, _X is an integer, and 14≤X≤30, wherein NX in the forward oligonucleotide chain and the reverse oligonucleotide The N _X in the nucleotide has the feature of reverse complementarity; complementary oligonucleotide double-stranded fragments are obtained by annealing;

c. Mix the CRISPR/Cas9 single transcription unit backbone vector with the complementary oligonucleotide double-stranded fragment obtained in step b, add BsaI endonuclease and T4 DNA ligase to the reaction system at the same time, and set "37°C restriction enzyme digestion" -16°C "ligation" cycle reaction to obtain a Cas9-gRNA recombinant vector specifically modified for the target site.

Specifically, the length of the specific target sequence in step a is 18-21 bp.

Preferably, the length of the specific target sequence in step a is 20bp.

Preferably, in step b, 18≤X≤21.

Preferably, a fusion PCR amplification strategy is applied in step c to obtain multiple gRNA transcription units tandem amplification products separated by ribozyme recognition cleavage sites (RZ cleavagesite), and cycle through "BsaI digestion-T4 DNA ligase ligase" cycle In a reaction manner, the BsaI-ccdB-BsaI unit is replaced, and the multi-gRNA transcription unit is cloned into the gRNA cloning and transcription unit to obtain a recombinant vector that can be specifically modified for multiple target sites.

The core unit of the present invention (Cas9 ORF-Poly A-RZ cleavage site-gRNA cloningscaffold-RZ cleavage site-RZ) can be connected to any PolII type promoter for specific transformation of host organisms and experimental needs, and Cas9 can be activated by different promoters -gRNA unit transcription (for example, CMV, hsP70, SV40, which are commonly used in animal cells, and CaMV35S, ZmUb1, AtUb10 and other promoters commonly used in plants, etc.), to realize the assembly of Cas9-gRNA splicing complex.

In the present invention, based on the CRISPR/Cas9 single transcription unit scaffold vector, after completing the construction of a specific Cas9-gRNA expression vector for transformation, under the condition of living cells, the Pol II promoter drives the "Cas9-Poly A-RZ site-gRNA cloning scaffold-RZ" site-RZ" is transcribed as an integral transcription unit to obtain single-stranded primary transcripts. Under the action of RZ, a single primary transcript undergoes self-cleavage at two RZ sites, respectively, to obtain a complete Cas9 protein expression box mRNA (including Poly A) and a gRNA transcription unit. In the cell system, the Cas9 protein expression box mRNA (containing Poly A) is further translated to obtain the Cas9 protein, and combined with the existing gRNA unit to form a functional Cas9-gRNA complex unit for targeted cleavage of the genome target site.

In the present invention, the complete guide RNA is formed by replacing the BsaI-ccdB-BsaI unit in the gRNA clone and transcription unit of the backbone vector with a 18-21 bp RNA fragment that can be complementary to the target fragment; the sequence of the RNA clone and transcription unit is Invariant: the sequence that does not include the BsaI-ccdB-BsaI unit is as in Seq ID No.3; the sequence that includes the BsaI-ccdB-BsaI unit is as described in Seq ID No.4) The backbone RNA fragment is in turn formed by a guide that can bind to the protospacer site gRNA, tracrRNA, crRNA chimera formed functional RNA similar to hairpin structure, and the backbone RNA fragment can be combined with Cas9 nuclease.

For a specific target gene, after determining the gRNA site (5'-N _X -NGG-3'; N represents any one of A, G, C, T, X is an integer, and 14≤X≤30(18 , 19, 20, and 21 are commonly used values)), according to the Cas9-gRNA recombinant vector construction method provided in the invention, the designed gRNA-specific target sequence (protospacer) "BsaI digestion-T4 DNA ligase ligase" cycle reaction In this way, the BsaI-ccdB-BsaI unit was replaced and cloned into the gRNA cloning and transcription unit to obtain a specific functional Cas9-gRNA recombinant vector.

In the present invention, a BsaI-ccdB-BsaI unit is fused to the 5' end of the gRNA clone transcription framework unit, which functions as a multi-cloning site to digest the CRISPR/Cas9 single transcription unit backbone vector, so as to clone the target gRAN-specific target sequence (protospacer). The key content of the present invention can be effectively achieved by replacing the BsaI-ccdB-BsaI unit with a restriction enzyme that can introduce a nick on the backbone vector of the present invention, and modifying the gRAN-specific target sequence cloning site accordingly.

In the present invention, a BsaI-ccdB-BsaI unit of 637 bp is fused to the 5' end of the gRNA transcription expression frame. The BsaI-ccdB-BsaI unit is used for cloning with a specific target sequence (protospacer) that recognizes the target gene. Through the synergistic action of BsaI endonuclease and T4 DNA ligase, the target site-specific Cas9-gRNA expression vector can be quickly and efficiently completed. When constructing a Cas9-gRNA recombinant vector for the specific modification of the target site, the specific complementary oligonucleotide duplex replaced the 637bp BsaI-ccdB-BsaI unit in the backbone vector and entered the CRISPR/Cas9 single transcription unit backbone vector.

In the process of constructing the Cas9-gRNA expression vector modified by the target site-specific sequence, the recombinant clones containing the correct Cas9-gRNA expression vector can be screened by transforming E. coli and bacterial screening, and colony PCR, plasmid digestion, sequence To identify the Cas9-gRNA recombinant expression vector for targeted modification of the genome of the target organism.

The ribozyme holoenzyme (RZ) used in the core unit of the present invention and its corresponding ribozyme recognition cleavage site (RZ cleavage site) need to be used together, but can have different variations, such as: 1) The original The RZ element in the present invention, but the nucleotide sequence of the RZ cleavage site in the present invention is substituted, deleted or one or more nucleotides are added, so that it can still be recognized and cut by the RZ element in the present invention; 2) can Replace the RZ element of the hammerhead ribozyme in the present invention with other types of ribozymes (eg: class I intron, RNaseP, hairpin ribozyme, hepatitis D ribozyme or VS ribozyme) , and correspondingly replace the RZ cleavage site in the present invention, so that it can be recognized and cut by the replaced ribozyme. These changes do not affect the core content of the present invention, that is: the Cas9 protein coding frame sequence (Cas9 ORF) and the gRNA cloning and transcription unit are recognized and cleaved by a specific ribozyme (RZ) through the ribozyme recognition cleavage site (RZ cleavage site (RZ cleavage). site) as a single transcription unit, so that the Cas9 ORF and the gRNA transcription unit can be co-transcribed by any Pol II type promoter.

By applying the fusion PCR amplification strategy, the tandem amplification products of multiple gRNA transcription units separated by the ribozyme recognition cleavage site (RZ cleavage site) can be obtained. By replacing the BsaI-ccdB-BsaI unit, this multi-gRNA transcription unit can be cloned into the gRNA cloning and transcription unit to obtain a Cas9-gRNA1-gRNA2-...-gRNAx recombinant vector that can be specifically modified for multiple target sites (Figure 5) .

In the present invention, the Cas9-gRNA recombinant vector constructed according to the present invention is transferred into plant cells through a variety of transformation methods mediated by protoplasts, gene guns and Agrobacterium, so that the transformed cells have both Cas9 nuclease protein and specific genomic targets. The gRNA unit of the sequence; under the combined action of the Cas9 nuclease protein and the gRNA unit, the targeted DNA double-strand of a specific genome target sequence is cut in a direction; and then under the action of the cell's endogenous DNA repair pathway, the target sequence NHEJ (nonhomologous end joining, Non-homologous end joining) or HR (homologous recombination, homologous recombination) directional modification results. When the CRISPR/Cas9 single transcription unit backbone vector of the present invention is applied in plants, resistance genes including kanamycin, hygromycin, basta and the like can be used to screen plant transformants, and the positive transformant cells or tissues ( Such as protoplast or callus) differentiation and regeneration to obtain regenerated plants containing targeted modification of the target site.

Beneficial effects of the present invention: The present invention provides an efficient CRISPR/Cas9 single transcription unit skeleton vector, which can effectively realize the coordinated transcription of Cas9 and gRNA units driven by Pol II type promoters, and is simple for a variety of eukaryotes. , Fast and efficient genome-directed genetic modification. In the backbone vector of the present invention, driven by a Pol II type promoter, the Cas9 nuclease protein expression frame and the gRNA transcription expression frame are in the same transcription unit, and 637bp of BsaI-ccdB- The BsaI unit, through the synergistic action of BsaI endonuclease and T4 DNA ligase, can quickly and efficiently complete the target site-specific Cas9-gRNA expression vector. The strategy that the Cas9 nuclease protein expression cassette and the gRNA transcription expression cassette are regulated by a unique Pol II promoter can effectively realize the coordinated expression of the Cas9 nuclease protein and the gRNA transcription unit, and improve the directional splicing efficiency of the CRSIPR/Cas9 system. By using common Pol II promoters for related animals and plants (such as CMV commonly used in animal cells and Ca MV35S, ZmUbi1 and other promoters commonly used in plants), the need to use species-specific U6, U3 and other small promoters in different species can be effectively avoided. The problem of RNA transcription promoters greatly expands the application scope of genome-directed genetic modification based on the CRSIPR/Cas9 system. At the same time, by further using different tissue-specific and inducible expression promoters, tissue-specific and inducible genome-directed genetic modification can be effectively achieved.

Description of drawings

Figure 1 Schematic diagram of the structure and working principle of the CRISPR/Cas9 single transcription unit designed in the present invention; Pol II promoter: Pol II type promoter; Cas9 ORF: Cas9 protein coding frame; RZ cleavage site: RZ recognition cleavage site; gRNA cloning scaffold : gRNA clone and transcription unit; transcription: transcription; translation: translation; Cas9protein: Cas9 protein; cleavage: shearing;

Figure 2 is a schematic diagram of a CRISPR/Cas9 single transcription unit expression vector specifically constructed and used for a specific rice endogenous gene locus in Examples 2 to 5 of the present invention. Among them: the specific Pol II type promoter used is the cauliflower mosaic virus 35S promoter (CaMV 35S promoter); in different embodiments, the gRNA cloning and transcription unit includes a 18-21 bp guide RNA unit that can specifically bind to the target site.

Figure 3 is a graph of the results of site-directed mutation detection of rice endogenous genes OsYSA, OsPDS, OSMPK2, and OsROC5 based on the CRISPR/Cas9 single transcription unit backbone vector of the present invention. Among them, lane 1 is the PCR amplification product of the corresponding rice gene, lanes 2 and 3 are the PCR amplification products of the wild-type and CRISPR/Cas9 expression vector transformation materials of different rice genes respectively digested by the corresponding restriction enzymes (Fig. 3a: OsYSA-gRNA1: SfiI; Figure 3b: OsYSA-gRNA2: EcoNI; Figure 3c: OsPDS-gRNA2: HindIII; Figure 3d: OsMPK2-gRNA: MscI; Figure 3e: OsROC5-gRNA: AhdI);

Figure 4 For different rice endogenous genes, the corresponding Cas9-gRNA expression vector was constructed based on the CRISPR/Cas9 single transcription unit backbone vector. After rice protoplasts were transformed, the PCR products were cloned and sequenced for the targeted modification target site. Among them, WT represents the wild-type gene sequence, M1 and M2 represent two independent directional modification results, "-" represents the sequence with deletion mutation, "+" represents the sequence with insertion mutation, and "-/+" after the sequence The numbers indicate the number of deleted or inserted nucleotides.

Figure 5. Schematic diagram of the tandem of multiple gRNA transcription units.

Figure 6 is a schematic diagram of the CRISPR/Cas9 single transcription unit expression vector constructed and used for two different sites of the rice endogenous gene OsYSA. Wherein: the specific Pol II type promoter used is the cauliflower mosaic virus 35S promoter (CaMV 35Spromoter); the OsYSA-gRNA1 and OsYSA-gRNA2 clones and transcription units respectively comprise 18-21bp guide RNA units that can specifically bind to the target site.

Figure 7 is a graph of the results of simultaneous site-directed mutation detection of two different sites of the rice endogenous gene OsYSA based on the CRISPR/Cas9 single transcription unit backbone vector of the present invention. Among them, lane 1 is the PCR amplification product of the corresponding OsYSA gene, lanes 2 and 3 are the results of directional modification induced by OsYSA-gRNA1 (SfiI restriction resistance band), and lanes 4 and 5 are the directional modification induced by OsYSA-gRNA2 Modification results (EcoNI digestion resistance band).

Figure 8 For the multi-gene locus of the rice endogenous gene OsYSA, a multi-gene locus knockout Cas9-gRNA expression vector was constructed based on the CRISPR/Cas9 single transcription unit backbone vector, and after the rice protoplasts were transformed, the PCR products were carried out for the targeted modification target site Clonal sequencing results. Among them, WT indicates the wild-type gene sequence, "-" indicates the sequence with deletion mutation, "+" indicates the sequence with insertion mutation, and the number after "-/+" indicates the number of deleted or inserted nucleotides.

Detailed ways

Example 1 Construction of CRISPR/Cas9 single transcription unit backbone vector

The present invention designs a CRISPR/Cas9 single transcription unit skeleton vector for genome engineering, the core unit of which is a Pol II type promoter (which can be realized by the scheme of AscI and SbfI double enzyme cutting the basic CRISPR/Cas9 single transcription unit skeleton vector) Replacement of different Pol II type promoters), Cas9 protein coding frame (including NLS sequence), Poly A sequence, ribozyme recognition site (RZ cleavage site), gRNA cloning and transcription unit (including BsaI-ccdB-BsaI unit), The ribozyme recognition site (RZ cleavage site) and the ribozyme (holoenzyme) (RiboZyme) are sequentially constituted. The structure and working principle of CRISPR/Cas9 single transcription unit are shown in Figure 1.

Optionally, the backbone vector also includes: left and right border sequences of T-DNA, and a single transcription unit driven by the Pol II promoter "Cas9-poly A-RZ cleavage site-gRNA cloning scaffold-RZ cleavage site-RZ" is located in all Between the left and right borders of the T-DNA; the left and right border sequences of the T-DNA also include a hygromycin resistance gene expression unit (the elements in sequence are: 2×CaMV35S promoter-hygromycin phosphotransferase ORF-CaMVpoly A” ; The replacement of different resistance gene ORFs can be realized by the scheme of AvrII, PacI double enzyme cutting the basic CRISPR/Cas9 single transcription unit backbone vector) as a plant transformant selection marker.

In order to realize the fast and efficient construction of a specific genomic target Cas9-gRNA expression vector, the CRISPR/Cas9 single transcription unit backbone vector of the present invention is integrated into the 637bp BsaI-ccdB-BsaI unit at the 5' end of the gRNA transcription expression unit, based on This design strategy, in the subsequent construction of the target Cas9-gRNA expression vector, only needs to mix the CRISPR/Cas9 single transcription unit backbone vector of the present invention, the annealed specific target sequence complementary oligonucleotide duplex in the construction system Fragment, BsaI endonuclease and T4 DNA ligase, and set up the cycle reaction of "37°C digestion -16°C ligation", the efficient construction of a specific Cas9-gRNA expression vector can be achieved. The vector can be constructed by conventional methods in the existing molecular cloning technology, and it should be noted that the above-mentioned element sequence is a unique part of the backbone plasmid vector, and it may also include some general structures of conventional vectors, which are not included in the present invention. More tired.

Based on Streptococcus pyogenes Cas9 nuclease protein-encoding gene (Streptococcus pyogenes Cas9, SpCas9), codon optimization (adding NLS signal at the 3' end), artificial synthesis of the complete ORF sequence of the Cas9 nuclease protein-encoding gene (including 3' NLS of the segment), the DNA sequence is shown in Seq ID No.2. Further, another 3 basic units are obtained by artificial synthesis: a, frag-A (Poly A+ ribozyme recognition site: shown in Seq ID No. 8); b, frag-B (BsaI-ccdB-BsaI unit: Seq ID No. 9); c, frag-B (gRNA transcription unit + ribozyme recognition site + ribozyme holoenzyme: Seq ID No. 10). By fusion PCR, the Cas9 nuclease protein ORF, frag-A, frag-B, and frag-C were fused in sequence, and SbfI and SacI restriction sites were added to the 5' and 3' ends of the fusion PCR product, respectively, to obtain 5013bp assembly unit. SbfI, SacI double digestion was performed on the vector backbone pTC097 (Cermak T, Starker CG, VoytasDF. Efficient design and assembly of custom TALENs using the Golden Gate platform. Methods Molecular Biology, 2005, 1239: 133-59.) and the 5013bp assembly unit, respectively , the target fragment was recovered, ligated and transformed, and the positive clones screened were confirmed by sequencing to complete the construction of the CRISPR/Cas9 single transcription unit backbone vector.

Example 2. Site-directed genetic modification of rice endogenous gene OsYSA based on CRISPR/Cas9 single transcription unit system

1. Target gRNA design of rice OsYSA gene and construction of Cas9-gRNA recombinant expression vector

Based on the rice OsYSA sequence (NCBI No. NM001057140) as the reference sequence, OsYSA-gRNA1 and OsYSA were designed according to the regions 365bp-387bp ( CCG CTTCGGCCGAGGTGGCGCGC, underlined as PAM structure) and 571bp-593bp ( CCT CATGAAGGTGCTCGTCGCGG, underlined as PAM structure). -gRNA2 (Table 1).

Table 1 Design, synthesis and detection information of rice OsYSA gene gRNA

According to the nucleic acid sequences of the designed OsYSA-gRNA1 and OsYSA-gRNA2 sites, the corresponding forward and reverse oligonucleotide chains were artificially synthesized, and the specific sequences were as follows (the uppercase base sequence represents the sequence of removing the PAM structure in the designed gRNA site; Lowercase base sequences represent cohesive end sequences complementary to the backbone vector):

OsYSA-gRNA1-F: cggaGCGCGCCACCTCGGCCGAAG

OsYSA-gRNA1-R: aaacCTTCGGCCGAGGTGGCGCGC

OsYSA-gRNA2-F: cggaCCGCGACGAGCACCTTCATG

OsYSA-gRNA2-R: aaacCATGAAGGTGCTCGTCGCGG

OsYSA-gRNA1-F/R and OsYSA-gRNA-2-F/R were mixed in equal proportions, boiled in a water bath for 10 min, and then cooled and annealed naturally to form double-stranded DNA with sticky ends, which was used as an insert for constructing a recombinant vector. Add CRISPR/Cas9 single transcription backbone vector, sticky-end insert, BsaI endonuclease, T4 DNA ligase to a 200uL PCR tube, set "37°C digestion-16°C ligation" for 10 cycles, and inactivate at 80°C After endo- and ligase, the reaction product was taken for E. coli transformation. Positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion. Finally, the corresponding Cas9-gRNA expression vectors were obtained by sequencing and were named pTX171::OsYSA-gRNA1 and pTX171::OsYSA-gRNA2 respectively.

2. The pTX171::OsYSA-gRNA1 and pTX171::OsYSA-gRNA2 expression vectors were transformed into rice protoplasts

Rice Nipponbare protoplasts were isolated and transformed into rice protoplasts with pTX171::OsYSA-gRNA1 and pTX171::OsYSA-gRNA2 expression vectors based on PEG method. The specific process of rice protoplast transformation Reference 1: Sheen, J. Signal transduction in maize and Arabidopsis mesophyllprotoplasts. Plant Physiology (2001) and Reference 2: Zahng, Y. et al. Transcriptionactivator-like effector nucleases enable efficient plant genomeengineering. Plant Physiology The experimental method disclosed in (2013).

3. Detection of results of targeted genetic modification of rice OsYSA gene

After rice protoplast transformation, cultured in the dark at 25°C for 48 hours, the transformed cells were collected, and the genomic DNA of rice protoplasts was extracted by CTAB method. The DNA was used as a template for PCR amplification and restriction endonuclease verification analysis (OsYSA-gRNA1, OsYSA -gRNA2 corresponds to SfiI and EcoNI, respectively, for enzyme digestion detection). For the specific method, refer to the experimental method disclosed in Zhang, Y. etal. Transcription activator-like effector nucleases enable efficient plantgenome engineering. Plant Physiology (2013). The experimental analysis results are shown in Figure 3a, 3b and Figure 4. The results in Figure 3a and 3b show that at the OsYSA-gRNA1 and OsYSA-gRNA2 sites, the rice endogenous sequence undergoes directional splicing mutation, and the mutation efficiency calculated by the software according to the band intensity is 28.9% and 34.8%, respectively; for Figure 3a The restriction enzyme resistance band in lane 3 of 3b was recovered, and TA was cloned and sequenced. The sequencing results are shown in Figure 4. The sequencing results showed that base mutations occurred at the OsYSA-gRNA1 and OsYSA-gRNA2 sites.

Example 3. Site-directed genetic modification of rice endogenous gene OsPDS based on CRISPR/Cas9 single transcription unit system

1. Target gRNA design of rice OsPDS gene and construction of pCas9-gRNA expression vector

OsPDS-gRNA2 was designed according to the rice OsPDS sequence (NCBI No. NM001055721) as the reference sequence and the region from 1290bp to 1312bp (GTTGGTCTTTGCTCCTGCAG AGG , underlined as PAM structure) (Table 2).

Table 2 Design, synthesis and detection information of rice OsPDS gene gRNA

According to the nucleic acid sequence of the designed OsPDS-gRNA2 site, the corresponding forward and reverse oligonucleotide chains were artificially synthesized, and the specific sequences were as follows (the uppercase base sequence represents the sequence in which the PAM structure was removed from the designed gRNA site; the lowercase base sequence represents the cohesive end sequence complementary to the backbone vector):

OsPDS-gRNA2-F: cggaGTTGGTCTTTGCTCCTGCAG

OsPDS-gRNA2-R: aaacCTGCAGGAGCAAAGACCAAC

OsPDS-gRNA2-F/R were mixed in equal proportions, bathed in boiling water for 10 min, and then cooled and annealed naturally to form double-stranded DNA with sticky ends, which was used as an insert for constructing a recombinant vector. Add CRISPR/Cas9 single transcription backbone vector, sticky-end insert, BsaI endonuclease, T4 DNA ligase to a 200uL PCR tube, set "37°C digestion-16°C ligation" for 10 cycles, and inactivate at 80°C After endo- and ligase, the reaction product was taken for E. coli transformation. The positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion, and finally the corresponding Cas9-gRNA expression vector was obtained by sequencing verification, which was named pTX171::OsPDS-gRNA2.

2. The pTX171::OsPDS-gRNA2 expression vector was transformed into rice protoplasts

Rice Nipponbare protoplasts were isolated and transformed into rice protoplasts with pTX171::OsPDS-gRNA2 expression vector based on PEG method. The specific process of rice protoplast transformation Reference 1: Sheen, J. Signaltransduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiology (2001) and Reference 2: Zhang, Y. et al. Transcription activator-like effector nucleasesenable efficient plant genome engineering. Plant Experimental method disclosed in Physiology (2013).

3. Detection of results of targeted genetic modification of rice OsPDS gene

After rice protoplast transformation, cultured in dark at 25°C for 48 hours, the transformed cells were collected, and the genomic DNA of rice protoplasts was extracted by CTAB method. The DNA was used as a template for PCR amplification and restriction endonuclease verification analysis (OsPDS-gRNA2 corresponds to HindIII). Enzyme digestion detection), the specific method reference: experimental method disclosed in Zahng, Y. et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology (2013). The experimental analysis results are shown in Figure 3c and Figure 4. The results in Figure 3c show that the rice endogenous sequence undergoes directional splicing mutation at the OsPDS-gRNA2 site, and the mutation efficiency calculated by the software according to the band intensity is 11.1%; for the restriction enzyme resistance band in lane 3 in Figure 3c Recovery, TA cloning and sequencing were performed. The sequencing results are shown in Figure 4. The sequencing results showed that a base mutation occurred at the OsPDS-gRNA2 site.

Example 4. Site-directed genetic modification of rice endogenous gene OsMPK2 based on CRISPR/Cas9 single transcription unit system

1. Target gRNA design of rice OsMPK2 gene and construction of Cas9-gRNA expression vector

OsMPK2-gRNA was designed according to the rice OsMPK2 sequence (NCBI No. NM001067563) as the reference sequence and the 65 bp-86 bp (GCGGCGGCCATGGCCATCA CGG , underlined as PAM structure) region (Table 3).

Table 3 Design, synthesis and detection information of rice OsMPK2 gene gRNA

According to the nucleic acid sequence of the designed OsMPK2-gRNA site, the corresponding forward and reverse oligonucleotide chains were artificially synthesized, and the specific sequences were as follows (the uppercase base sequence represents the sequence in which the PAM structure was removed from the designed gRNA site; the lowercase base sequence represents the cohesive end sequence complementary to the backbone vector):

OsMPK2-gRNA-F: cggaGCGGCGGCCATGGCCATCA

OsMPK2-gRNA-R: aaacTGATGGCCATGGCCCGCCGC

OsMPK2-gRNA-F/R were mixed in equal proportions, bathed in boiling water for 10 min, and then cooled and annealed naturally to form double-stranded DNA with sticky ends, which was used as an insert for constructing a recombinant vector. Add CRISPR/Cas9 single transcription backbone vector, sticky-end insert, BsaI endonuclease, T4 DNA ligase to a 200uL PCR tube, set "37°C digestion-16°C ligation" for 10 cycles, and inactivate at 80°C After endo- and ligase, the reaction product was taken for E. coli transformation. Positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion, and finally the corresponding Cas9-gRNA expression vector was obtained by sequencing and verified, and named pTX171::OsMPK2-gRNA.

2. The pTX171::OsMPK2-gRNA expression vector was transformed into rice protoplasts

Rice Nipponbare protoplasts were isolated and transformed into rice protoplasts with pTX171::OsMPK2-gRNA expression vector based on PEG method. The specific process of rice protoplast transformation Reference 1: Sheen, J. Signaltransduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiology (2001) and Reference 2: Zhang, Y. et al. Transcription activator-like effector nucleasesenable efficient plant genome engineering. Plant Experimental method disclosed in Physiology (2013).

3. Detection of results of targeted genetic modification of rice OsMPK2 gene

After rice protoplast transformation, cultured in the dark at 25°C for 48 hours, the transformed cells were collected, and the genomic DNA of rice protoplasts was extracted by CTAB method. The DNA was used as a template for PCR amplification and restriction endonuclease verification analysis (OsMPK2-gRNA corresponds to MscI Enzyme digestion detection), the specific method reference: experimental method disclosed in Zahng, Y. et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology (2013). The experimental analysis results are shown in Figure 3d and Figure 4: Figure 3d shows that at the OsMPK2-gRNA site, the rice endogenous sequence undergoes directional splicing mutation, and the mutation efficiency calculated by the software according to the band intensity is 10.5%; for Figure 3d The restriction enzyme-resistant band in lane 3 was recovered, and TA cloned and sequenced. The sequencing results are shown in Figure 4. The sequencing results showed that a base mutation occurred at the OsMPK2-gRNA site.

Example 5. Site-directed genetic modification of rice endogenous gene OsROC5 based on CRISPR/Cas9 single transcription unit system

1. Target gRNA design of rice OsROC5 gene and construction of Cas9-gRNA expression vector

OsROC5-gRNA was designed according to the rice OsROC5 sequence (NCBI No. NM001054253) as the reference sequence and the region from 647 bp to 669 bp (GCGGAGAACGACAGCCGGTC GGG , underlined as PAM structure) (Table 4).

Table 4 Design, synthesis and detection information of rice OsROC5 gene gRNA

According to the nucleic acid sequence of the designed OsROC5-gRNA site, the corresponding forward and reverse oligonucleotide chains were artificially synthesized, and the specific sequences were as follows (the uppercase base sequence represents the sequence in which the PAM structure was removed from the designed gRNA site; the lowercase base sequence represents the cohesive end sequence complementary to the backbone vector):

OsROC5-gRNA-F: cggaGCGGAGAACGACAGCCGGTC

OsROC5-gRNA-R: aaacGACCGGCTGTCGTTCTCCGC

OsROC5-gRNA-F/R were mixed in equal proportions, bathed in boiling water for 10 min, and then cooled and annealed naturally to form double-stranded DNA with sticky ends, which was used as an insert for constructing a recombinant vector. Add CRISPR/Cas9 single transcription backbone vector, sticky-end insert, BsaI endonuclease, T4 DNA ligase to a 200uL PCR tube, set "37°C digestion-16°C ligation" for 10 cycles, and inactivate at 80°C After endo- and ligase, the reaction product was taken for E. coli transformation. The positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion, and finally the corresponding Cas9-gRNA expression vector was obtained by sequencing verification, which was named pTX171::OsROC5-gRNA.

2. The pTX171::OsROC5-gRNA expression vector was transformed into rice protoplasts

Rice Nipponbare protoplasts were isolated and transformed into rice protoplasts with pTX171::OsROC5-gRNA expression vector based on PEG method. The specific process of rice protoplast transformation Reference 1: Sheen, J. Signaltransduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiology (2001) and Reference 2: Zhang, Y. et al. Transcription activator-like effector nucleasesenable efficient plant genome engineering. Plant Experimental method disclosed in Physiology (2013).

3. Detection of results of targeted genetic modification of rice OsROC5 gene

After rice protoplast transformation, the cells were cultured in the dark at 25°C for 48 hours, and the transformed cells were collected. The genomic DNA of rice protoplasts was extracted by CTAB method, and the DNA was used as a template for PCR amplification and restriction endonuclease verification analysis (OsROC5-gRNA corresponds to AhdI Enzyme digestion detection), the specific method reference: experimental method disclosed in Zahng, Y. et al. Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiology (2013). The experimental analysis results are shown in Figure 3e and Figure 4. The results in Figure 3e show that at the OsROC5-gRNA site, the rice endogenous sequence undergoes directional splicing mutation, and the mutation efficiency calculated by the software according to the band intensity is 26.7%; for the restriction enzyme resistance band in lane 3 in Figure 3e Recovery, TA cloning and sequencing were performed. The sequencing results are shown in Figure 4. The sequencing results showed that a base mutation occurred at the OsROC5-gRNA site.

Example 6. Simultaneous site-directed genetic modification of rice endogenous gene OsYSA at multiple sites based on CRISPR/Cas9 single transcription unit system

1. Design of multi-site target gRNA of rice OsYSA gene and construction of Cas9-gRNA recombinant expression vector

Based on the rice OsYSA sequence (NCBI No. NM001057140) as the reference sequence, OsYSA-gRNA1 and OsYSA were designed according to the regions 365bp-387bp ( CCG CTTCGGCCGAGGTGGCGCGC, underlined as PAM structure) and 571bp-593bp ( CCT CATGAAGGTGCTCGTCGCGG, underlined as PAM structure). -gRNA2 (Table 5).

Table 5 Design, synthesis and detection information of rice OsYSA gene gRNA

According to the nucleic acid sequences of the designed OsYSA-gRNA1 and OsYSA-gRNA2 sites, the corresponding forward and reverse oligonucleotide chains were artificially synthesized, and the specific sequences were as follows (the uppercase base sequence represents the sequence of removing the PAM structure in the designed gRNA site; Lowercase base sequences represent cohesive end sequences complementary to the backbone vector):

OsYSA-gRNA1-F: cggaGCGCGCCACCTCGGCCGAAG

OsYSA-gRNA1-R: aaacCTTCGGCCGAGGTGGCGCGC

OsYSA-gRNA2-F: cggaCCGCGACGAGCACCTTCATG

OsYSA-gRNA2-R: aaacCATGAAGGTGCTCGTCGCGG

OsYSA-gRNA1-F/R and OsYSA-gRNA-2-F/R were mixed in equal proportions, boiled in water for 10 min, and then cooled and annealed naturally to form double-stranded DNA with sticky ends, which was used as an insert for constructing a recombinant vector. Add CRISPR/Cas9 single transcription backbone vector (Figure 5), two sticky end inserts, BsaI endonuclease, and T4 DNA ligase to a 200uL PCR tube, and set "37°C digestion-16°C ligation" for 10 cycles of reaction , after treatment at 80°C to inactivate the endonuclease and ligase, the reaction product was taken for E. coli transformation. Positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion. Finally, the corresponding Cas9-gRNA expression vector was obtained by sequencing and verified, and named pTX171::OsYSA-gRNA1-gRNA2 (Figure 6).

2. The pTX171::OsYSA-gRNA1-gRNA2 expression vector was transformed into rice protoplasts

Rice Nipponbare protoplasts were isolated and transformed into rice protoplasts with pTX171::OsYSA-gRNA1-gRNA2 expression vector based on PEG method. The specific process of rice protoplast transformation Reference 1: Sheen, J. Signaltransduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiology (2001) and Reference 2: Zhang, Y. et al. Transcription activator-like effector nucleasesenable efficient plant genome engineering. Plant Experimental method disclosed in Physiology (2013).

3. Detection of the results of simultaneous directed genetic modification of rice OsYSA gene at multiple loci

After rice protoplast transformation, cultured in the dark at 25°C for 48 hours, the transformed cells were collected, and the genomic DNA of rice protoplasts was extracted by CTAB method. The DNA was used as a template for PCR amplification and restriction endonuclease verification analysis (OsYSA-gRNA1, OsYSA -gRNA2 corresponds to SfiI and EcoNI, respectively, for enzyme digestion detection). For the specific method, refer to the experimental method disclosed in Zahng, Y. etal. Transcription activator-like effector nucleases enable efficient plantgenome engineering. Plant Physiology (2013). The experimental analysis results are shown in Figure 7 and Figure 8. The results in Figure 7 show that at the sites of OsYSA-gRNA1 and OsYSA-gRNA2, the endogenous sequences of rice have undergone directional splicing mutations; the resistant bands in lanes 2, 3, 4, and 5 in Figure 7 were recovered respectively, and TA was performed. Cloning and sequencing, the sequencing results are shown in Figure 8, and the sequencing results show that mutations have occurred at the OsYSA-gRNA1 and OsYSA-gRNA2 sites.

Claims (9)

1. The single transcriptional units skeleton carrier of 1.CRISPR/Cas9, Cas9 and guide RNA core cell are regulated and controled by a promoter Transcription, the promoter be II type promoter of Pol, from 5 ' to the 3 ' direction of core cell is followed successively by Cas9 ORF- Poly A-RZ cleavage site-gRNA cloning scaffold-RZ cleavage site-RZ, and gRNA Cloning scaffold is at least one, is RZ cleavage site between gRNA cloning scaffold；Wherein, Cas9ORF, that is, Cas9 encoding histone frame, RZ cleavage site, that is, ribozyme identify cleavage site, gRNA cloning Scaffold, that is, gRNA clone and transcriptional units, RZ, that is, ribozyme holoenzyme.
2. 2. skeleton carrier as described in claim 1, it is characterised in that: gRNA is cloned and transcriptional units are 1~5.
3. 3. skeleton carrier as described in claim 1, it is characterised in that: Cas9 encoding histone frame also includes nuclear localization signal NLS Sequence has the amino acid sequence as shown in Seq ID No.1.
4. 4. skeleton carrier as claimed in claim 1, it is characterised in that: gRNA clone and transcriptional units have as in Seq ID No.3 Shown in nucleotide sequence.
5. 5. skeleton carrier as claimed in claim 1, it is characterised in that: the ribozyme of the ribozyme holoenzyme and corresponding identification identifies cutting Site is used cooperatively in the following manner:

   Ribozyme holoenzyme is replaced, while corresponding replacement ribozyme identifies cleavage site, and replaced ribozyme identifies cleavage Ribozyme holoenzyme identification after point is replaced is cut.
6. 6. skeleton carrier as claimed in claim 5, it is characterised in that: the ribozyme holoenzyme is hammerhead ribozyme, coding Nucleotide sequence sequence as shown in Seq ID No.6.
7. 7. skeleton carrier as claimed in claim 6, it is characterised in that: the ribozyme identification cleavage site is by hammerhead ribozyme Identification cutting, nucleotide sequence is as shown in Seq ID No.5.
8. 8. skeleton carrier as described in claim 1, it is characterised in that: the Cas9ORF-poly A-RZ cleavage Site-gRNA cloning scaffold-RZ cleavage site-RZ has the nucleotides sequence as shown in Seq ID No.7 Column.
9. 9. skeleton carrier as described in any one of claims 1 to 8, it is characterised in that: the II type promoter of Pol is flower coconut palm It is dish mosaic virus 35 S promoter CaMV35S, corn Ubi1 promoter ZmUbi1, arabidopsis Ubi10 promoter AtUbi10, big and small Cellular virus CMV, Heat shock protein 70 promoter hsP70 or 40 promoter SV40 of vacuolating virus of monkey.