阅读说明：本技术 一种基于CRISPR-Cas9技术的基因编辑系统及应用 (Gene editing system based on CRISPR-Cas9 technology and application ) 是由 童英 卢俊南 张明虹 陈立志 梁兴祥 姚永超 秦莉 陈小平 于 2019-01-11 设计创作，主要内容包括：本发明提供了一种基于CRISPR-Cas9技术的基因编辑系统及其应用,以内源U6启动子依赖型载体系统为基础,在表达载体的Cas9表达盒内加入负筛选标记,并将包含同源臂的拯救载体上的抗药筛选标记转移至表达载体上,减少拯救载体的基础大小,扩大其容量,可介导在人疟原虫中敲入长达6.3kb外源基因片段,且获得的重组疟原虫不含抗药筛选标记和Cas9蛋白表达质粒的残留,可用同一药筛标记进行连续基因编辑,性能稳定,高效简洁,功能强大,具有广阔的应用前景和巨大的市场价值。(The invention provides a gene editing system based on CRISPR-Cas9 technology and application thereof, based on an endogenous U6promoter dependent vector system, a negative screening marker is added into a Cas9 expression cassette of an expression vector, and an anti-drug screening marker on a rescue vector containing a homologous arm is transferred onto the expression vector, so that the basic size of the rescue vector is reduced, the capacity of the rescue vector is enlarged, an exogenous gene segment with the length of 6.3kb can be knocked into human plasmodium by mediating, the obtained recombinant plasmodium does not contain the anti-drug screening marker and the residue of a Cas9 protein expression plasmid, continuous gene editing can be carried out by using the same drug screening marker, and the gene editing system is stable in performance, high-efficiency, simple, powerful, and has wide application prospect and great market value.)

1. A gene editing system based on CRISPR-Cas9 technology is characterized in that the system is endogenous U6promoter dependent and comprises an expression vector and a rescue vector;

the expression vector comprises a drug resistance screening marker, an sgRNA expression cassette and a Cas9 expression cassette, and the Cas9 expression cassette comprises a negative screening marker;

the rescue vector comprises a homologous arm insertion site and an exogenous gene fragment insertion site.

2. The system of claim 1, wherein the drug-resistance screening marker comprises any one or a combination of at least two of HDHFR, BSD, NEO, PAC, or YDHODH, preferably BSD;

preferably, the negative selection marker comprises yFCU and/or HSV-TK, preferably yFCU.

3. A host cell comprising the system of claim 1or 2;

preferably, the host cell comprises any one or a combination of at least two of plasmodium falciparum, plasmodium vivax, plasmodium knowlesi, plasmodium malariae or plasmodium ovale, preferably plasmodium falciparum.

4. A method of plasmodium gene editing using the system of claim 1or 2, comprising the steps of:

(1) constructing an expression vector and a rescue vector according to claim 1or 2;

(2) screening a target sequence according to a target gene and connecting the target sequence to an expression vector;

(3) cloning exogenous genes and homologous arms derived from target genes onto a rescue vector;

(4) preparing the expression vector in the step (2) and the rescue vector in the step (3) into DNA-loaded RBC;

(5) adding plasmodium schizonts to the DNA-loaded RBCs prepared in step (4);

(6) carrying out drug resistance screening and negative screening to obtain recombinant plasmodium;

(7) performing monoclonality on the recombinant plasmodium obtained in the step (6), and performing expression vector residue detection and drug sensitivity test on different monoclonals;

(8) and (4) carrying out gene editing again on the plasmodium by using the same drug screening marker according to the operation method of the steps (1) to (6).

5. The method of claim 4, wherein the Plasmodium comprises any one or a combination of at least two of Plasmodium falciparum strains Pf3D7, PfNF54, PfFCR3 or PfDd2, preferably Pf3D 7;

preferably, the target gene in the step (2) comprises a red internal phase non-essential gene Pf47 or P230P, preferably a red internal phase non-essential gene Pf 47.

6. The method according to claim 4 or 5, wherein the foreign gene of step (3) comprises R787OD and/or an antigenic protein gene, preferably R787 OD.

7. The method according to any one of claims 4 to 6, wherein the method for preparing DNA-loadedRBCs in step (4) comprises: replicating the vector in escherichia coli, extracting and purifying, and electrically transferring to human red blood cells to prepare DNA-loaded RBC;

preferably, the preparation method of the schizont in the step (5) comprises the following steps: and (3) carrying out gelatin enrichment on the Pf3D7 insect strain cultured in vitro to obtain a schizont.

8. The method according to any one of claims 4 to 7, comprising in particular the steps of:

(1) constructing an expression vector and a rescue vector according to claim 1or 2;

(2) screening a target sequence according to a target gene Pf47 and connecting the target sequence to an expression vector;

(3) cloning a foreign gene R787OD and a homology arm derived from a target gene Pf47 onto a rescue vector;

(4) copying the expression vector in the step (2) and the rescue vector in the step (3) in escherichia coli, extracting, purifying, electrically transferring to human red blood cells, and preparing into DNA-loaded RBC;

(5) performing gelatin enrichment on the Pf3D7 insect strain cultured in vitro to obtain a schizont, and adding the schizont into the DNA-loaded RBC prepared in the step (4);

(6) carrying out drug resistance screening, PCR sequencing identification and exogenous gene expression analysis, and then carrying out negative screening;

(7) performing monoclonality on the recombinant plasmodium obtained in the step (6), and performing expression vector residue detection and drug sensitivity test on different monoclonals;

(8) and (4) carrying out gene editing again on the plasmodium by using the same drug screening marker according to the operation method of the steps (1) to (6).

9. A recombinant plasmodium produced by the method of any one of claims 4 to 8.

10. Use of the system of claim 1or 2, the host cell of claim 3, the method of any one of claims 4-8, or the recombinant plasmodium of claim 9 for plasmodium gene large fragment knock-in and/or for designing a tumor vaccine.

Technical Field

The invention belongs to the technical field of biology, and relates to a gene editing system based on a CRISPR-Cas9 technology and application thereof.

Background

The CRISPR/Cas9 technology has the advantages of simple design, multiple sites, high efficiency, good specificity, short time consumption and the like, and is the hottest gene editing technology at present, the main elements of the CRISPR/Cas9system comprise a target gene targeting sNA (single guide RNA) and a Cas9 nuclease, the action principle of the system is that the Cas9 nuclease cuts at a specific site of a target gene under the guidance of the sgRNA to generate a gap, cells can repair the gap in various ways after the gap is generated, and the like mainly comprises classical non-homologous end join (NHEJ) repair and homologous recombination (homologous recombination) HR repair, and the like.

Among Plasmodium falciparum (Pf), two different CRISPR/Cas9 technical systems are currently available for selection, an endogenous U6 promoter-dependent (U6promoter dependent, U6Pd) type and an exogenous T7 promoter-dependent (T7promoter dependent, T7Pd) type. The U6Pd system adopts a dual plasmid design (Ghorbal, M., et al (2014) 'Genome editing in the human macrolararia parasite plasmid using the CRISPR-Cas9system', Nat Biotechnol 32(8): 819. 821.), one vector is a Cas9 protein expression vector which carries a Cas9 protein expression frame and a drug resistance screening marker expression frame, and the size of the plasmid is 11096 bp; the other vector is an sgRNA expression vector and homologous repair template, and carries an sgRNA expression frame, a negative screening marker, a drug resistance screening marker and a homologous arm, wherein the sgRNA is expressed by a Pf endogenous U6promoter, and the size of a plasmid without the homologous arm is 9233 bp. The T7Pd system also adopts a double plasmid system (Wagner J.C., et al (2014) 'effective CRISPR-Cas9-mediated genome editing in plasmid falciparum.' Nat Methods 11(9):915 918), one plasmid is a Cas9 protein expression vector which carries a Cas9 protein expression cassette, an sgRNA expression cassette and an anti-drug screening marker expression cassette, wherein the sgRNA transcription is started by a T7promoter, and the sgRNA cannot be transcribed autonomously due to the fact that T7RNA polymerase is absent in Plasmodium; the other plasmid is a T7RNA polymerase expression vector and a homologous arm carrying vector, and simultaneously carries another drug resistance screening marker.

The disadvantages of both systems are: 1. the gene-edited worm contains the residue of the drug resistance screening marker and the Cas9 protein expression vector. The drug resistance screening markers available in Pf are limited at present, and only five kinds of drug resistance screening markers are selected from hdhfr, bsd (fibrinolysin Sdaminase), neo (neomycin phosphotransferase), pac (puromycin-N-acetyl transferase), ydhdodh (yeast dihydroorotate dehydrogenase), and the drugs are expensive and are not commercialized individually; if multiple genes of plasmodium need to be edited, the two gene editing systems can be difficult to realize multiple rounds of gene editing of Pf due to insufficient drug screening marks; while the residue of the Cas9 protein expression vector will create a potential risk of off-target gene editing; 2. the size of the plasmid base carrying the homology arms for homology repair is large, and large fragments are difficult to load for knock-in operation.

Therefore, the Cas9/sgRNA expression vector is improved and optimized, the knock-in of a gene large fragment, the removal of a drug-resistant screening marker and continuous gene editing are realized, and the method has wide application prospect and great market value.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides a gene editing system based on a CRISPR-Cas9 technology and application thereof, wherein the system is based on an endogenous U6promoter dependent vector system, a negative screening marker is added on an expression vector, a drug-resistant screening marker on a rescue vector containing a homology arm is transferred onto the expression vector, experimental conditions are optimized and searched, a gene large fragment is finally and successfully knocked in, a recombinant insect strain which is free of drug-resistant markers and Cas9 protein expression vector residues and can be continuously subjected to gene editing by using the same drug screening marker is obtained, and the gene editing system has wide application prospect and great market value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a gene editing system based on a CRISPR-Cas9 technology, wherein the system is endogenous U6promoter dependent and comprises an expression vector and a rescue vector;

the expression vector comprises a drug resistance screening marker, an sgRNA expression cassette and a Cas9 expression cassette, and the Cas9 expression cassette comprises a negative screening marker;

the rescue vector comprises a homologous arm insertion site and an exogenous gene fragment insertion site, and does not contain a drug screen marker.

In the invention, in long-term scientific research practice, the inventor deeply researches the advantages and disadvantages of a CRISPR/Cas9system in plasmodium gene editing, improves a U6Pd type CRISPR/Cas9system in order to solve the problems of knocking-in Cas9 plasmid and drug-resistant marker residue, realizes the insertion and continuous gene editing of a large gene fragment, finds through a large amount of experiments, integrates sgRNA expression cassettes originally dispersed in two vectors and a Cas9 expression cassette into the same vector to construct a new expression vector, and the vector carries a drug-resistant screening marker and a negative screen marker besides the sgRNA and Cas9 expression cassettes; the other vector is a homologous arm vector which does not carry any drug screening marker. The action principle of the system is as follows: the Cas9/sgRNA complex can induce DNA gaps that would be fatal in the absence of a homologous template due to the lack of an effective end-joining repair mechanism in plasmodium, which would necessitate the presence of a homologous arm vector in order for the plasmodium to survive rescue. If only Cas9 and sgRNA expression vectors are obtained by plasmodium, the insect strain will die because Cas9/sgRNA is sheared and cannot be repaired; if the plasmodium obtains only the homologous arm vector, the insect strain is killed because no drug-resistant screening marker exists; if the plasmodium obtains the two vectors, but correct homologous recombination repair does not occur, the strain will die due to the shearing of Cas9/sgRNA, unless Cas9 and sgRNA are not normally expressed, so the plasmodium can survive and be enriched under the positive screening drug screening only if the two vectors are simultaneously obtained and correct homologous recombination repair occurs, but the obtained live insect contains a drug resistance screening marker and the residue of the Cas9 protein expression vector. Because a small amount of plasmid-lost worms are generated in the plasmodium proliferation process, worms without drug-resistant screening markers and residual Cas9 protein expression vectors can be enriched by removing the positive screen drugs and adding the negative screen drugs for culture.

The system has the advantages that: 1. the use of medicine sieve marks is reduced; 2. the basic size of the homologous arm vector is reduced, and the function of loading larger gene segments is realized; 3. the negative screen marker realizes the purpose of removing the drug resistance screening marker and the residue of the Cas9 protein expression vector; 4. the same drug screen marker can be used for continuous gene editing.

Preferably, the drug resistance screening marker comprises any one or a combination of at least two of HDHFR, BSD, NEO, PAC or YDHODH, preferably BSD.

The drug resistance screening markers currently available in Pf are limited, and only five choices are hdhfr, bsd (cytosin Sdaminase), neo (neomycin phosphotransferase), pac (puromycin-N-acetyl transferase), and ydhod (yeast dihydroorotate dehydrogenase).

Preferably, the negative selection marker comprises yFCU and/or HSV-TK, preferably yFCU.

The expression product of the Saccharomyces cerevisiae derived negative selection gene yFCU (yeast cell deaminase and uracil phosphoribosyl transferase) is a dual functional chimeric protein of cytosine deaminase and uracil phosphoribosyltransferase, which catalyzes the non-toxic prodrug 5-Fluorocytosine (5-Fc) to become toxic 5-Fluorouracil (5-FU). Then, 5-FU is further metabolized to 5-fluoro-2 '-deoxyuridine-5' -monophosphate (5-FdUMP), and 5-FdUMP inhibits the activity of thymine synthase, resulting in the inability to synthesize Thymidine Triphosphate (TTP), the absence of TTP pools, and thus the inhibition of efficient DNA synthesis. Therefore, if the recombinant insect strain contains a plasmid containing the yfcu gene, the insect with the plasmid will die because the DNA synthesis is inhibited after the prodrug 5-Fc is added.

In a second aspect, the present invention provides a host cell comprising the system of the first aspect.

Preferably, the host cell comprises any one of plasmodium falciparum, plasmodium vivax, plasmodium knowlesi, plasmodium malariae, plasmodium ovale or a combination of at least two of them, preferably plasmodium falciparum.

In a third aspect, the present invention provides a method of plasmodium gene editing using the system according to the first aspect, comprising the steps of:

(1) constructing an expression vector and a rescue vector according to the first aspect;

(2) screening a target sequence according to a target gene and connecting the target sequence to an expression vector;

(3) cloning exogenous genes and homologous arms derived from target genes onto a rescue vector;

(4) preparing the expression vector in the step (2) and the rescue vector in the step (3) into DNA-loaded RBC;

(5) adding plasmodium schizonts to the DNA-loaded RBCs prepared in step (4);

(6) carrying out drug resistance screening to obtain recombinant plasmodium, carrying out PCR sequencing identification and exogenous gene expression analysis, carrying out negative screening to obtain plasmodium with lost drug resistance screening marker, negative screening marker and Cas9 coding sequence,

(7) and (4) performing monoclonality on the recombinant plasmodium obtained in the step (6), performing expression vector residue detection and drug sensitivity test on different monoclonals, and detecting drug screening marks and Cas9 protein expression plasmid residues.

(8) And (4) carrying out gene editing again on the plasmodium by using the same drug screening marker according to the operation method of the steps (1) to (6).

Preferably, the plasmodium comprises any one or a combination of at least two of plasmodium falciparum strains Pf3D7, PfNF54, PfFCR3 or PfDd2, preferably Pf3D 7;

preferably, the target gene in the step (2) comprises a red internal phase non-essential gene Pf47 or P230P, preferably a red internal phase non-essential gene Pf 47.

Preferably, the target sequence of step (2) is SEQ ID NO. 1.

SEQ ID NO.1 is as follows:

AACTACAGTTGGCTTAACAT.

preferably, the exogenous gene of step (3) comprises R787OD and/or an antigenic gene, preferably R787 OD.

The R787OD recombinant protein is constructed based on an artificial PfEMP1R29var-V5-GFP-TM-ATS (Melcher, M., et al (2010). "Identification of a roll for the PfEMP1semi-conserved head structure in protein trafficking to the surface of plasmid engineered red cells" Microbiol 12(10): 1446. sup. 1462.), after the N-terminal domain of the R29var protein, "Age I site-GGPSG linker-PstI site" is added, after the V5 tag, a SacI site is added, after GFP, "SacI site-GGPSG linker-NUTR I4614 bp" is added, the size of the recombinant protein is expressed by the entire PbEF 1 α 5, 35's DT 3, 86' expressed by the whole size of the recombinant protein.

The invention inserts the recombinant artificial plasmodium falciparum surface protein 1 expression cassette (ORF length is 4.6kb) with the length of 6.3kb into plasmodium to verify the large fragment knock-in potential of the plasmid system. Plasmodium falciparum surface protein 1(Plasmodium falciparum erythrocyte membrane protein 1, PfEMP1) is currently the most extensively studied virulence factor of Plasmodium falciparum. After the plasmodium enters parasitic erythrocytes, the PfEMP1 is placed on the surfaces of the erythrocytes to change the structures of the erythrocyte skeletons and the cell membrane surfaces and combine with different vascular endothelial receptors, so that the erythrocytes infected with the plasmodium are gathered in blood vessels of different tissues, and the killing effect of the spleen of a human body is avoided; by utilizing the characteristic that PfEMP1 can be transported to the surface of red blood cells, some important structural domains of PfEMP1 are fused with exogenous gene fragments to construct recombinant artificial PfEMP1, so that the exogenous gene fragments are displayed on the surface of the red blood cells, and the strategy has important value in basic research and vaccine design.

Preferably, the method for preparing DNA-loaded RBCs in step (4) comprises: the vector is replicated in colon bacillus, extracted and purified, and then electrically transferred to human red blood cells to prepare the DNA-loaded RBC.

Preferably, the preparation method of the schizont in the step (5) comprises the following steps: and (3) carrying out gelatin enrichment on the Pf3D7 insect strain cultured in vitro to obtain a schizont.

The detailed steps are as follows:

the first step is as follows: constructing a universal Cas9/sgRNA expression vector and a rescue vector;

the universal Cas9/sgRNA expression vector includes: a sgRNA expression cassette controlled by P.falciparum U6 small nuclear RNA polymerase 3 promoter (U6snRNA polymerase III promoter, 5' U6, Gene ID: PF3D7_134110) promoter sequence and 3' noncoding region (3 ' U6) for guiding Cas9 nuclease to target a target site, wherein BtgZI cleavage site is included, and sgRNA sequence of target Gene can be inserted at the site;

an expression cassette for the Blasticidin S resistance Gene (Blasticidin-S deaminase, BSD, EC number:3.5.4.23) controlled by the promoter sequence (5 ' CAM) of the P.falciparum calmodulin Gene (calmod. mu. L in, CAM, Gene ID: PF3D 7-1434200) and the 3' noncoding region (3 ' HRP2) of the P.falciparum HRPII Gene (histide-rich protein II, HRPII, Gene ID: PF3D 7-0831800) for resistance selection of Gene editing protozoa;

a yfcu and Cas9 expression cassette controlled by p.falciparum heat shock protein 86(heat shock protein 86, Gene ID: PF3D7_0708400) promoter (5 ' hsp86) and 3' non-coding sequence (3 ' PbDT) of Plasmodium breviceri plasmidium berghei bifunctional dihydrofolate reductase thymidine synthase (DHFR-TS, Gene ID: pbaa _0719300), the yfcu Gene being a negative sieve marker for screening of Plasmodium falciparum which does not contain a drug resistance marker and a Cas9 protein expression plasmid, Cas9 being a nuclease which cleaves target DNA;

in addition, it also includes an Ampicillin (Ampicillin, Amp) expression frame, and is used for screening positive clone in Escherichia coli (such as DH5 α, X L-10, Stbl3 and NEB Stable plasmid DNA clone strain) and maintaining the stability of said plasmid in Escherichia coli, and the basic skeleton of plasmid DNA and plasmid DNA replication origin (ColE1origin) sequence in Escherichia coli, etc.

The universal rescue vector mainly comprises a reporter Gene GFP and a renalla lucifera coding sequence (3 'PbDT) controlled by the 5' promoter of P.falciparum translational elongation factor (5 'PfEf 1 α, elongation factor 1-alpha, Gene ID: PF3D7_1357000) and the 3' non-coding sequence of Plasmodium boidinii (DHFR-TS, Gene ID: PBANKANKA _0719300), a plasmid for cloning Escherichia coli strains (5 'PfEf 3834, elongation factor 1-alpha), a plasmid for cloning Escherichia coli strains (5' PfEf1 α, 3 'Pbelf 1 α, a plasmid for cloning Escherichia coli strains (5' PfEf), a plasmid for cloning Escherichia coli strains (3 'PfEf), a plasmid for cloning Escherichia coli strains (5' PfEf, 3 'Pb5-alpha), a plasmid for cloning strains (3, 5' PfEf), a plasmid for cloning strains of Escherichia coli strains (3, 5's resistance, 5's plasmid for cloning strains, a plasmid for cloning strains of Escherichia coli strains of.

The second step is that: screening a Cas9/sgRNA target sequence, connecting the target sequence to a universal Cas9/sgRNA expression vector, and constructing a Cas9/sgRNA expression vector capable of targeting a target gene, wherein the target gene can be a plasmodium erythrocytic stage non-essential gene or a plasmodium toxicity related gene;

the third step: cloning a homologous arm and an exogenous gene fragment from a target gene onto a universal rescue vector; the exogenous gene comprises an antigen gene, a therapeutic agent gene, an immunomodulator gene or a peptide gene;

the fourth step: copying the two vectors in escherichia coli, extracting, purifying, electrically transferring into human red blood cells, and preparing into DNA-loaded RBC;

the fifth step: performing gelatin enrichment on a plasmidium falciparum 3D7(Pf3D7) insect strain cultured in vitro to obtain a schizont, and adding the schizont into DNA-loaded RBC;

and a sixth step: screening the medicine to obtain recombinant plasmodium;

the seventh step: performing PCR identification and exogenous gene expression analysis on the recombinant plasmodium;

eighth step: negative screening is carried out on the recombinant plasmodium;

the ninth step: monoclonality is carried out on the plasmodium after negative screening;

the tenth step: and (3) carrying out drug screening marking and Cas9 protein expression plasmid residue detection on the recombinant plasmodium monoclonal after negative screening.

The eleventh step: the same drug screen marker is used for re-gene editing of plasmodium by the method.

Preferably, the method for plasmodium gene editing, which uses the system of the first aspect, specifically comprises the following steps:

(1) constructing an expression vector and a rescue vector according to the first aspect;

(2) screening a target sequence according to a target gene Pf47 and connecting the target sequence to an expression vector;

(3) cloning a foreign gene R787OD and a homology arm derived from a target gene Pf47 onto a rescue vector;

(4) copying the expression vector in the step (2) and the rescue vector in the step (3) in escherichia coli, extracting, purifying, electrically transferring to human red blood cells, and preparing into DNA-loaded RBC;

(5) performing gelatin enrichment on the Pf3D7 insect strain cultured in vitro to obtain a schizont, and adding the schizont into the DNA-loaded RBC prepared in the step (4);

(6) carrying out drug resistance screening, PCR sequencing identification and exogenous gene expression analysis, and carrying out negative screening to obtain plasmodium with lost drug resistance screening markers, negative screening markers and Cas9 coding sequences;

(7) carrying out expression vector residue detection and drug sensitivity test on the recombinant plasmodium after single cloning in the step (6), and detecting drug screening marks and Cas9 protein expression plasmid residue;

(8) and (4) carrying out gene editing again on the plasmodium by using the same drug screening marker according to the operation method of the steps (1) to (6).

In a fourth aspect, the present invention provides a use of the system according to the first aspect or the method according to the third aspect for plasmodium gene large fragment knock-in and/or tumor vaccine design.

The existing research results show that plasmodium infection can efficiently activate the inherent immunity of an organism and tumor antigen specificity CT L s, antagonize a tumor immunosuppressive microenvironment and inhibit tumor angiogenesis, the fever caused by plasmodium infection can kill tumor cells and has long action duration, the plasmodium can express large exogenous genes, can contain and express a plurality of exogenous genes, and the expression level of the exogenous genes is high, so that the tumor antigen genes or the immunomodulator genes or the combination of the tumor antigen genes and the immunomodulator genes are knocked into the plasmodium to construct a tumor vaccine taking the plasmodium as a carrier, and the plasmodium vaccine has important application value in the treatment or prevention of tumors.

The knocking-in is to knock out a section of target gene and insert a new exogenous gene.

Compared with the prior art, the invention has the following beneficial effects:

the gene editing system provided by the invention is based on an endogenous U6 promoter-dependent vector system, a negative screening marker is added into a Cas9 expression cassette of an expression vector, and a drug-resistant screening marker on a rescue vector containing a homologous arm is transferred onto the expression vector, so that the basic size of the rescue vector is reduced, the capacity of the rescue vector is enlarged, an exogenous gene segment with the length of 6.3kb can be knocked into a human plasmodium through mediation, the obtained recombinant plasmodium does not contain the drug-resistant screening marker and the residue of a Cas9 protein expression plasmid, the same drug screening marker can be used for continuous gene editing, and the gene editing system is stable in performance, high-efficiency, simple and powerful in function.

Drawings

FIG. 1 shows the structure and sgRNA insertion site of Cas9/sgRNA expression vector pCBS-yfcu;

FIG. 2 shows the structure and cleavage site of rescue vector pARM-BtgZI (GFP/RUC) BtgZI, MCs are homologous arm insertion sites, BtgZI cleavage site is exogenous gene fragment insertion site;

FIG. 3 is a diagram of the structure of the pCBS-yfcu-pf47 plasmid;

FIG. 4 is a diagram showing the construction of the plasmid pARM-R787ODki-pf 47;

FIG. 5(A) shows the principles of knock-in and PCR identification of the R787OD fragment, L H and RH: the 5 'and 3' homology arms; P1, P2, P3, P4 represent PCR primers;

FIG. 5(B) shows the result of PCR detection; wt: wild-type pf3D7 insect strain; ki: a plasmodium strain into which the R787OD fragment knocks;

FIGS. 6(A) and 6(B) are the results of RT-PCR detection of transcription and protein expression of the target gene in R787OD transgenic Plasmodium, wt: wild-type pf3D7 insect strain; ki: a plasmodium strain into which the R787OD fragment knocks; -RT: a control without reverse transcriptase during reverse transcription, which is used for detecting whether DNA remains in RNA; + RT: adding reverse transcriptase during reverse transcription;

FIG. 6(C) shows the results of WesternBlot assay of transcription and protein expression of the target gene in R787OD transgenic Plasmodium, with GAPDH as the internal reference;

FIG. 7 shows the expression of the target gene in R787OD transgenic Plasmodium in erythrocytes, and Cy3 and Cy3 labeled goat anti-mouse secondary antibody (red fluorescence); BF, bright field; wt: wild-type pf3D7 insect strain; ki: a plasmodium strain into which the R787OD fragment knocks; scale bar 10 μm;

FIG. 8(A) is the PCR detection result of BSD and Cas9 encoding genes of the PCR detection of free plasmids in different monoclonals of R787OD transgenic Plasmodium negative screen; bulk: transgenic plasmodium before negative screening, Clone1, 2, 3, 4: the R787OD transgenic plasmodium monoclone after negative screening;

FIG. 8(B) is a graph of the integration of the R787OD fragment in the minus-screened insect strain, wt: wild-type pf3D7 insect strain, Clone 1: the R787OD transgenic plasmodium monoclone after negative screening;

FIG. 9 shows the expression of target gene in erythrocyte of R787OD transgenic Plasmodium after negative screening. Cy3, Cy3 labeled goat anti-mouse secondary antibody (red fluorescence); BF: bright field; wt: wild-type pf3D7 insect strain; ki-ep-: the R787OD transgenic plasmodium monoclone strain after negative screening; scale bar 10 μm;

fig. 10 is the killing effect of BSD on R787OD transgenic plasmodium after negative screening, wt: wild-type pf3D7 insect strain; ki-ep-: the R787OD transgenic plasmodium monoclonals after negative screening.

FIG. 11(A) shows the principle of typing-in the PfNT1 gene N-terminal GFP tag of R787OD transgenic Plasmodium and the principle of PCR identification, L H and RH: 5 'homology arm and 3' homology arm; P1, P2, P3, P4 represent PCR primers;

FIG. 11(B) shows the result of PCR detection; wt: wild-type pf3D7 or R787OD insect strain; ki: PfNT1 gene N-end knockin GFP-tagged Plasmodium;

fig. 11(C) fluorescent microscope observation of the knock-in GFP tag at the N-terminus of PfNT1 gene of R787OD transgenic plasmodium, BF: bright field.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.