WO2020143125A1 - Système d'édition de gènes basé sur la technologie crispr-cas9 et son utilisation - Google Patents

Système d'édition de gènes basé sur la technologie crispr-cas9 et son utilisation Download PDF

Info

Publication number
WO2020143125A1
WO2020143125A1 PCT/CN2019/082198 CN2019082198W WO2020143125A1 WO 2020143125 A1 WO2020143125 A1 WO 2020143125A1 CN 2019082198 W CN2019082198 W CN 2019082198W WO 2020143125 A1 WO2020143125 A1 WO 2020143125A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasmodium
gene
vector
cas9
expression
Prior art date
Application number
PCT/CN2019/082198
Other languages
English (en)
Chinese (zh)
Inventor
童英
卢俊南
张明虹
陈立志
梁兴祥
姚永超
秦莉
陈小平
Original Assignee
广州中科蓝华生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州中科蓝华生物科技有限公司 filed Critical 广州中科蓝华生物科技有限公司
Publication of WO2020143125A1 publication Critical patent/WO2020143125A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor
    • C12N1/105—Protozoal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application belongs to the field of biotechnology and relates to a gene editing system and application based on CRISPR-Cas9 technology.
  • Gene editing is a technique that uses artificial nucleases or "molecular scissors" to insert, delete, or replace DNA, including the first-generation ZFN zinc finger nuclease technology, the second-generation TALEN technology, and the third-generation CRISPR/Cas9 technology .
  • CRISPR/Cas9 technology has the advantages of simple design, multiple sites, high efficiency, good specificity, and short time consumption. It is currently the hottest gene editing technology.
  • the main elements of the CRISPR/Cas9 system are sgRNA (single guide RNA) targeting the target gene and Cas9 nuclease. The principle of this system is that under the guidance of sgRNA, Cas9 nuclease cuts at specific sites of the target gene, resulting in a gap.
  • the cell can be repaired in various ways, including classic non-homologous end (NHEJ) repair and homologous recombination (HR) repair.
  • NHEJ non-homologous end
  • HR homologous recombination
  • NHEJ repair often occurs. This repair process will result in the insertion or deletion of DNA, causing frameshift mutations, resulting in gene inactivation.
  • homologous recombination repair can occur to achieve the right Precise editing of target genes, such as introducing point mutations or inserting gene fragments; Plasmodium lacks a non-homologous end repair mechanism, and its DNA gap repair almost completely relies on homologous end repair.
  • U6promoter dependent, U6Pd endogenous U6 promoter dependent
  • T7promoter dependent, T7Pd exogenous T7 promoter dependent
  • One vector is Cas9 protein expression vector, carrying Cas9 protein expression box and drug resistance screening marker expression box, plasmid size is 11096bp; the other vector is sgRNA expression vector and homologous repair template, carrying sgRNA expression box, negative selection marker, resistance
  • the drug selection marker and the homology arm, sgRNA is expressed by the Pf endogenous U6 promoter, and the size of the plasmid without the homology arm is 9233bp.
  • the T7Pd system also uses a dual plasmid system (Wagner JC, et.al. (2014).
  • one plasmid is Cas9 Protein expression vector, carrying Cas9 protein expression box, sgRNA expression box and drug resistance screening marker expression box, in which the transcription of sgRNA is initiated by the T7 promoter. Since there is no T7 RNA polymerase in the malaria parasite, sgRNA cannot be transcribed autonomously; the other plasmid is The T7RNA polymerase expression vector and the homology arm carrying vector also carry another drug resistance screening marker.
  • the disadvantages of these two systems are: 1.
  • the gene-edited insects contain drug resistance screening markers and the residue of Cas9 protein expression vector.
  • the available drug screening markers in Pf are very limited. Only five options are hdhfr, bsd (blasticidin S deaminase), neo (neomycin phosphotransferase), pac (puromycin-N-acetyltransferase), ydhodh (yeast dihydroorotate dehydrogenase).
  • the drugs are expensive, and some have not been commercialized; if multiple genes of Plasmodium are required to be edited, the above two gene editing systems may be difficult to realize multiple rounds of gene editing of Pf due to insufficient drug screening markers; and Cas9
  • the residue of the protein expression vector will create a potential risk of off-target gene editing; 2.
  • the base size of the plasmid carrying the homology arm for homology repair is large, and it is difficult to load large fragments for knock-in operation.
  • the present application provides a gene editing system and application based on CRISPR-Cas9 technology, which is based on the endogenous U6 promoter-dependent vector system and adds negative screening to the expression vector Marker, and transfer the drug-resistant screening marker on the rescue vector containing the homology arm to the expression vector, optimize the experimental conditions and verify, and finally successfully knock in the gene large fragment to obtain the drug-free marker and no Cas9 protein expression vector residue 2.
  • CRISPR-Cas9 technology is based on the endogenous U6 promoter-dependent vector system and adds negative screening to the expression vector Marker, and transfer the drug-resistant screening marker on the rescue vector containing the homology arm to the expression vector, optimize the experimental conditions and verify, and finally successfully knock in the gene large fragment to obtain the drug-free marker and no Cas9 protein expression vector residue 2.
  • Recombinant insect strains that can be used for continuous gene editing with the same drug sieve marker have broad application prospects and huge market value.
  • this application provides a gene editing system based on CRISPR-Cas9 technology
  • the system is endogenous U6 promoter dependent, including expression vectors and rescue vectors;
  • the expression vector includes a drug resistance screening marker, an sgRNA expression cassette, and a Cas9 expression cassette, and the Cas9 expression cassette includes a negative screening marker;
  • the rescue vector includes a homology arm insertion site and a foreign gene fragment insertion site, and does not contain a drug sieve marker.
  • the applicant has in-depth research on the advantages and disadvantages of the CRISPR/Cas9 system in malaria parasite gene editing in order to solve the problem of knocking in the Cas9 plasmid and drug resistance marker residues, and realize the insertion of large gene fragments and continuous genes Editor, improved the U6Pd type CRISPR/Cas9 system, and found through a lot of experiments that the sgRNA expression cassette originally dispersed in two vectors and the Cas9 expression cassette were integrated into the same vector to construct a new expression vector.
  • the vector except sgRNA In addition to the Cas9 expression cassette, it also carries drug resistance screening markers and negative sieve markers; the other vector is a homologous arm vector, which does not carry any drug sieve markers.
  • Cas9/sgRNA complex can induce a DNA gap.
  • this DNA gap will be fatal and must be Only with the presence of homologous arm vectors can Plasmodium be rescued and survived.
  • Plasmodium only obtains cas9 and sgRNA expression vectors, the strain will die due to Cas9/sgRNA cleavage and cannot be repaired; if Plasmodium only obtains homologous arm vectors, then the strain will have no drug resistance The screening marker is killed; if the malaria parasite obtains the above two vectors, but the correct homologous recombination repair does not occur, then the strain will also die due to Cas9/sgRNA cleavage, unless Cas9 and sgRNA are not normally expressed, Therefore, Plasmodium can only survive at the same time when two vectors are obtained and correct homologous recombination repair can be survived and enriched.
  • the live worms obtained at this time contain drug resistance screening markers and the residue of Cas9 protein expression vector.
  • a small amount of insects with lost plasmids will be generated during the propagation of Plasmodium. Therefore, by removing the positive sieve drugs and adding negative sieve drugs for cultivation, the insects that do not contain the drug resistance screening marker and the Cas9 protein expression vector can be enriched.
  • the drug resistance screening marker includes any one or a combination of at least two of HDHFR, BSD, NEO, PAC, or YDHODH, preferably BSD.
  • hdhfr blasticidin S deaminase
  • neo neomycin phosphotransferase
  • pac puromycin-N-acetyltransferase
  • ydhodh yeast dihydroorotate dehydrogenase
  • the negative selection marker includes yFCU and/or HSV-TK, preferably yFCU.
  • Saccharomyces cerevisiae-derived negative selection gene yFCU yeast cytosine deaminase and urine bosyl transferase
  • yFCU yeast cytosine deaminase and urine bosyl transferase
  • cytosine deaminase and uracil phosphoribosyl transferase bifunctional chimeric protein this protein can catalyze a non-toxic precursor
  • the drug 5-Fluorocytosine (5-Fc) turns it into a toxic 5-Fluorouracil (5-FU).
  • 5-FU can be further metabolized to 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP), and 5-FdUMP can inhibit the activity of thymine synthase, resulting in thymidine triphosphate (TTP) cannot be synthesized, and the TTP pool is missing, thereby inhibiting the effective synthesis of DNA. Therefore, if there is a plasmid containing the yfcu gene in the recombinant insect strain, after adding the prodrug 5-Fc, the insect with the plasmid will die because of the inhibition of DNA synthesis.
  • the present application provides a host cell, the host cell comprising the system of the first aspect.
  • the host cell includes any one of Plasmodium falciparum, Plasmodium vivax, Plasmodium noveli, Plasmodium falciparum, or a combination of at least two Plasmodium ovale, preferably Plasmodium falciparum.
  • the present application provides a method for gene editing of Plasmodium using the system as described in the first aspect, including the following steps:
  • the Plasmodium includes any one or a combination of at least two of P. falciparum strains Pf3D7, PfNF54, PfFCR3 or PfDd2, preferably Pf3D7;
  • the target gene in step (2) includes a non-essential gene Pf47 or P230P in the erythrocytic phase, preferably a non-essential gene Pf47 in the erythrocytic phase.
  • the target sequence in step (2) is SEQ ID NO.1.
  • SEQ ID NO.1 is as follows:
  • the foreign gene in step (3) includes R787OD and/or antigen gene, preferably R787OD.
  • the R787OD recombinant protein is constructed based on artificial PfEMP1R29var-V5-GFP-TM-ATS (Melcher, M., et.al. (2010). "Identification of the petroleum for PfEMP1 semi-conserved head structure in protein of traffic trafficking to the surface of Plasmorum. infected cells.
  • Plasmodium falciparum surface protein 1 (Plasmodium falciparum erythrocyte membrane protein 1, PfEMP1) is currently the most thoroughly studied virulence factor of Plasmodium falciparum.
  • PfEMP1 is placed on the surface of erythrocytes to change the structure of erythrocyte skeleton and cell membrane surface, combined with different vascular endothelial receptors, resulting in the aggregation of erythrocytes infected with Plasmodium in blood vessels of different tissues, escaping Killing effect; using the feature that PfEMP1 can be transported to the surface of red blood cells, some important domains of PfEMP1 are fused with foreign gene fragments to construct recombinant artificial PfEMP1, so that the foreign gene fragments are displayed on the surface of red blood cells. Both basic research and vaccine design have important value.
  • the method for preparing the DNA-loaded RBC described in step (4) is as follows: the vector is replicated in E. coli, extracted and purified, and transferred to human erythrocytes to prepare the DNA-loaded RBC.
  • the preparation method of the schizont in step (5) is: enriching the Pf3D7 worm strain cultured in vitro to obtain schizont.
  • the first step construct a universal Cas9/sgRNA expression vector and rescue vector
  • the universal Cas9/sgRNA expression vector includes: a P. falciparum U6 small nuclear RNA polymerase 3 promoter (U6snRNA polymerase III promoter, 5'U6, Gene ID: PF3D7_134110) promoter sequence and a 3'non-coding region (3' U6)
  • the controlled sgRNA expression frame is used to guide the Cas9 nuclease to the target site, which contains the BtgZI cleavage site, and the sgRNA sequence targeting the target gene can be inserted at this site;
  • a promoter sequence (5'CAM) of P.falciparum gene (calmodulin, cam, Gene: ID: PF3D7_1434200) and 3'of P. falciparum HRPII gene (histidine-rich protein II, HRPII, Gene ID: PF3D7_0831800)
  • the expression box of the blasticidin S resistance gene (Blasticidin-S deaminase, BSD, EC number: 3.5.4.23) controlled by the non-coding region (3'HRP2) is used for resistance screening of gene editing Plasmodium;
  • a P. falciparum heat shock protein 86 (heat Shock protein 86, Gene ID: PF3D7_0708400) promoter (5'hsp86) and Plasmodium berghei Plasmodium berghei bifunctional dihydrofolate reductase thymidine synthase (bifunctional dihydrofolate reductase -thymidylate synthase, DHFR-TS, Gene ID: PBANKA_0719300), the 3'end non-coding sequence (3'PbDT) controls the yfcu and Cas9 expression boxes, the yfcu gene is a negative sieve marker, used for drug resistance-free markers and Cas9 protein Screening for residual Plasmodium in expression plasmids, Cas9 is a nuclease that cleaves target DNA;
  • E. coli also includes an ampicillin resistance gene (Ampicillin, Amp) expression box, which is used to screen and maintain positive clones in E. coli (such as DH5 ⁇ , XL-10, Stbl3, and NEB Stable plasmid DNA cloning strains). Stability in E. coli; and the basic skeleton of plasmid DNA and the sequence of the origin of the replication of plasmid DNA in E. coli (ColE1origin) sequence, etc.
  • Amp ampicillin resistance gene
  • the universal rescue vector mainly includes: a 5'promoter (5'PfEf1 ⁇ , elongation factor 1-alpha, Gene ID: PF3D7_1357000) of P.falciparum's translation elongation factor and Plasmodium berghei bifunctional dihydrogen
  • the folate reductase thymidine synthase (bifunctional dihydrofolate reductase-thymidylate synthase, DHFR-TS, Gene ID: PBANKA_0719300) 3'non-coding sequence (3'PbDT) controlled reporter gene GFP and renilla luciferase expression box (GFP/RUC ), at both ends of GFP and renilla luciferase coding sequences with BtgZI cleavage sites, BtgZI digestion can replace the reporter gene GFP/RUC with other genes.
  • a 5'promoter 5'PfEf1 ⁇ , elongation factor 1-al
  • cleavage sites There are multiple cleavage sites (MCs) upstream of 5'-PfEf1 ⁇ and downstream of 3'-PbDT for insertion of 5'and 3'homology arms; an ampicillin resistance gene (Ampicillin, Amp) expression box, Used for screening positive clones in E. coli (such as DH5 ⁇ , XL-10, Stbl3 and NEB Stable plasmid DNA cloning strains) and maintaining the stability of the plasmid in E. coli; and the basic skeleton of plasmid DNA and in the large intestine Plasmid DNA replication origin (ColElorigin) sequence in Bacillus.
  • E. coli such as DH5 ⁇ , XL-10, Stbl3 and NEB Stable plasmid DNA cloning strains
  • Step 2 Screen the Cas9/sgRNA target sequence, connect the target sequence to the universal Cas9/sgRNA expression vector, and construct a Cas9/sgRNA expression vector that can target the target gene.
  • Genes or genes related to Plasmodium toxicity are also known in the art.
  • the third step clone the homologous arms and foreign gene fragments from the target gene into a universal rescue vector;
  • the foreign genes include antigen gene, therapeutic agent gene, immunomodulator gene or peptide gene;
  • Step 4 Copy and extract the above two vectors in E. coli, then transfer them to human red blood cells to prepare DNA-loaded RBC;
  • Step 5 Enrich Plasmodium falciparum 3D7 (Pf3D7) worm strains cultured in vitro to obtain schizonts, and add the schizonts to the DNA-loaded RBC;
  • Step 6 Obtain recombinant Plasmodium by drug screen
  • Step 7 PCR identification and analysis of foreign gene expression of recombinant Plasmodium
  • Step 8 Negative screening of recombinant Plasmodium
  • the ninth step monoclonalize the malaria parasite after negative screening
  • Step 10 Carry out drug screening and detection of the residue of Cas9 protein expression plasmid on the recombinant plasmodium monoclonals after negative screening.
  • the eleventh step using the above method to use the same drug sieve marker to regenerate the malaria parasite.
  • a method for gene editing of Plasmodium using the system described in the first aspect specifically includes the following steps:
  • step (2) and the rescue vector of step (3) are replicated in E. coli, extracted and purified, and transferred to human red blood cells to prepare DNA-loaded RBC;
  • step (6) After the recombinant Plasmodium of step (6) is monoclonalized, the expression vector residue detection and drug sensitivity test are carried out to detect the drug sieve marker and Cas9 protein expression plasmid residue;
  • the present application provides a system as described in the first aspect or the method as described in the third aspect for the application of a large fragment of Plasmodium gene knock-in and/or design of a tumor vaccine.
  • Plasmodium infection can effectively activate the body's innate immunity and tumor antigen-specific CTLs, antagonize the tumor immunosuppression microenvironment, inhibit tumor angiogenesis; fever caused by Plasmodium infection can kill tumor cells, and the effect continues Long time; Plasmodium can express large foreign genes, can accommodate and express multiple foreign genes, and the expression level of foreign genes is high, therefore, the tumor antigen gene or immunomodulator gene or The combination of the two to construct a tumor vaccine using Plasmodium as a carrier will have important application value in the treatment or prevention of tumors.
  • the knock-in is to knock out a target gene and insert a new foreign gene.
  • the gene editing system provided in this application is based on the endogenous U6 promoter-dependent vector system, a negative selection marker is added to the Cas9 expression cassette of the expression vector, and the drug resistance selection marker on the rescue vector containing the homology arm is transferred to On the expression vector, reducing the basic size of the rescue vector and expanding its capacity can mediate the knock-in of 6.3 kb foreign gene fragments in human Plasmodium, and the obtained recombinant Plasmodium does not contain drug resistance screening markers and Cas9 protein expression
  • the residue of the plasmid can be used for continuous gene editing with the same drug sieve marker. The performance is stable, efficient and concise, and powerful.
  • 1 is the structure of the Cas9/sgRNA expression vector pCBS-yfcu and the sgRNA insertion site of the embodiment of the present application;
  • Figure 2 is the structure and cleavage site of rescue vector pARM-BtgZI (GFP/RUC) BtgZI, MCs is the insertion site of the homology arm, and BtgZI cleavage site is the insertion site of the foreign gene fragment;
  • GFP/RUC rescue vector pARM-BtgZI
  • Figure 3 is a pCBS-yfcu-pf47 plasmid structure diagram
  • Figure 4 is a pARM-R787ODki-pf47 plasmid structure diagram
  • Fig. 5(A) is the principle of R787OD fragment knock-in and PCR identification, LH and RH: 5'homology arm and 3'homology arm; P1, P2, P3, P4 represent PCR primers;
  • Figure 5(B) is the result of PCR detection; wt: wild-type pf3D7 strain; ki: Plasmodium strain with R787OD fragment knocked in;
  • Figure 6(A) and Figure 6(B) are RT-PCR detection results of target gene transcription and protein expression in R787OD transgenic Plasmodium, wt: wild-type pf3D7 strain; ki: Plasmodium falciparum with knock-in of R787OD fragment Strain; -RT: control without adding reverse transcriptase during reverse transcription, used to detect whether there is DNA residue in RNA; +RT: adding reverse transcriptase during reverse transcription; actin as internal reference;
  • Figure 6(C) is the Western Blot test results of target gene transcription and protein expression in R787OD transgenic Plasmodium, GAPDH as internal reference;
  • Figure 8(A) shows the PCR detection results of BSD and Cas9 encoding genes of the free plasmids in different monoclonals of negative screening of R787OD transgenic Plasmodium; original: transgenic Plasmodium before negative screening, clones 1, 2, 3, 4 : R787OD transgenic Plasmodium monoclonal after negative screening;
  • Fig. 8(B) is the detection of the integration of R787OD fragment in the strain after negative screening
  • wt wild-type pf3D7 strain
  • clone 1 clone of R787OD transgenic Plasmodium after negative screening
  • Fig. 9 shows the expression of target genes in R787OD transgenic Plasmodium in red blood cells.
  • Cy3, Cy3 labeled goat anti-mouse secondary antibody (red fluorescence); BF: bright field; wt: wild-type pf3D7 strain; ki-ep-: R787OD transgenic Plasmodium monoclonal strain after negative screening; scale 10 ⁇ m;
  • Figure 10 is the killing effect of BSD on R787OD transgenic Plasmodium after negative screening, wt: wild-type pf3D7 strain; ki-ep-: R787OD transgenic Plasmodium monoclonal strain after negative screening;
  • Fig. 11(A) is the principle of PfNT1 gene Nf-terminal GFP tag knock-in and PCR identification principle of R787OD transgenic Plasmodium.
  • LH and RH 5'homology arm and 3'homology arm; P1, P2, P3, P4 represent PCR Primer
  • Figure 11(B) is the result of PCR detection; wt: wild-type pf3D7 or R787OD strain; ki: Plasmodium with PfNT1 gene knocked into the GFP tag at the N-terminus;
  • Fig. 11(C) Fluorescence microscope observation result of knocking in the GFP tag at the N-terminus of PfNT1 gene of R787OD transgenic Plasmodium, BF: bright field.
  • the yeast-derived negative selection gene yfcu and cas9 gene are merged into the same expression cassette, The two are connected by a 2A peptide sequence (SEQ ID NO. 2: GGTTCGGGAGAGGGCAGAGGATCCCTGCTAACATGCGGTGATGTCGAGGAGAATCCTGGCCCAGAATCGCTCGAG), and the yfcu-2A fusion gene is directly inserted into the XhoI site in front of the Cas9 gene ORF.
  • the specific construction process is as follows:
  • the first step pCC4 plasmid (Maier, AG., et.al. (2006). "Negative selection” using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted targeted gene deletion by double crossover recombination. 121.) as a template, PCR amplification of the ORF fragment of the yfcu gene, the reaction system is shown in Table 1:
  • Reaction procedure 98 °C 30s; 98 °C 10s, 50 °C 15s, 60 °C 2min, 33 cycles; 60 °C 7min; 16 °C storage; 1% agarose gel electrophoresis to recover the target fragment.
  • PCR primers and sequence are as follows:
  • sequence of 160817Ryfcu is SEQ ID NO. 3:
  • sequence of 160817Fyfcu is SEQ ID NO.4:
  • Step 2 Overlap PCR to obtain the coding segment of the yfcu-2A fusion gene.
  • the first step is to anneal and synthesize the coding segment of the 2A peptide.
  • the reaction system is shown in Table 2:
  • Reaction procedure 98 °C 30s; 98 °C 10s, 50 °C 30s, 60 °C 1min; 4 °C stored for future use.
  • the primer sequence is as follows:
  • the yfcu gene and 2A are connected by overlap PCR, and the ORF amplified fragment of the yfcu gene is directly added to the 2A annealing extension reaction system.
  • the reaction procedure is: 98 °C 10s, 50 °C 30s, 60 °C 2min, 3 cycles; 60 °C 7min; save at 4°C for use; add 1 ⁇ L of forward amplification primer 160817Fyfcu gene, and then carry out PCR amplification: 98 °C 10s, 50 °C 15s, 60 °C 2min, 29 cycles; 60 °C 7min; 16 °C,
  • the target fragment was recovered by 1% agarose gel electrophoresis.
  • the third step XhoI single digestion to obtain the pCBS plasmid framework, the digestion reaction system and procedures are operated according to the Xho I manual. Incubate at 37°C for 2h, and recover by 1% agarose gel electrophoresis.
  • the rapid cloning method (Vazyme, C112-02) was ligated to construct the target vector.
  • the amount of reaction system, plasmid framework and insert used was calculated according to the kit instructions.
  • the ligation product was transformed into XL10 competent cells (Vazyme, C503-03), and the transformation was carried out according to the instructions of the competent state.
  • the bacteria grow out of the ampicillin-resistant plate, pick a single colony into a centrifuge tube containing 5mL LB medium, 37 °C, 220rpm shaking culture 10-11h, save the strain (500 ⁇ L bacterial solution, add 500 ⁇ L 30% glycerol, After mixing and cryopreservation at -80°C), the remaining bacterial solution is used for plasmid extraction using a plasmid mini-extraction kit, and the operation steps are carried out according to the instructions of the plasmid extraction kit.
  • the plasmid was sequenced and identified.
  • the sequencing primers and sequences are as follows:
  • the pCBS-yfcu plasmid successfully constructed through the above steps is about 13.4kb in size, and its structure is shown in Figure 1, including: a P.falciparum U6 small nuclear RNA polymerase 3 promoter (U6 snRNA polymerase III promoter, 5'U6 , Gene ID: PF3D7_134110) promoter sequence and 3'non-coding region (3'U6) controlled sgRNA expression frame, used to guide Cas9 nuclease to target sites. It contains the BtgZI restriction site, where the sgRNA sequence targeting the target gene can be inserted; a promoter sequence (5'CAM) consisting of the P.
  • a P.falciparum U6 small nuclear RNA polymerase 3 promoter U6 snRNA polymerase III promoter, 5'U6 , Gene ID: PF3D7_134110
  • 3'U6 controlled sgRNA expression frame used to guide Cas9 nuclease to target sites. It contains
  • falciparum calmodulin gene (calmodulin, cam, Gene ID: PF3D7_1434200) and P. falciparum HRPII gene (histidine-rich protein II, HRPII, Gene ID: PF3D7_0831800) 3'non-coding region (3'HRP2) controlled blasticidin S resistance gene (Blasticidin-S deaminase, BSD, EC number:3.5.4.23) expression box, used for resistance screening of gene editing Plasmodium; a promoter of P.falciparum heat shock protein 86 (heat Shock protein 86, Gene ID: PF3D7_0708400) (5'hsp86) and Yfcu controlled by the 3'non-coding sequence (3'PbDT) of Plasmodium berghei Plasmodium berghei bifunctional dihydrofolate reductase thymidine synthase (bifunctional dihydrofolate reductase-thymidylate synthase
  • the structure of the rescue vector is shown in Figure 2. Its main components are: a 5'promoter (5'PfEf1 ⁇ , elongation factor 1-alpha, Gene ID: PF3D7_1357000) of P.falciparum's translation elongation factor, and Mouser Plasmodium berghei Plasmodium berghei bifunctional dihydrofolate reductase thymidine synthase (bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS, Gene ID: PBANKA_0719300) 3'end of the non-coding sequence (3'PbDT) controlled by the reporter gene GFP And renilla luciferase expression frame (GFP/RUC), with BtgZI cleavage sites at both ends of GFP and renilla luciferase coding sequences, the reporter gene GFP/RUC can be replaced with other genes by BtgZI digestion.
  • cleavage sites There are multiple cleavage sites (MCs) upstream of 5'-PfEf1 ⁇ and downstream of 3'-PbDT for insertion of 5'and 3'homology arms; an ampicillin resistance gene (Ampicillin, Amp) expression box, For screening positive clones in Escherichia coli (such as DH5 ⁇ , XL-10, Stbl3 and NEB Stable plasmid DNA cloning strains) and maintaining the stability of the plasmid in E. coli; and the basic skeleton of plasmid DNA and in the large intestine
  • the plasmid DNA replication origin (ColE1origin) sequence in Bacillus, the specific construction process is as follows:
  • Step 1 Amplify the AmpR-ori fragment from the commercial T vector (pMDTM19-T).
  • the PCR amplification reaction system is the same as Table 1.
  • the reaction conditions were: 98 °C 30s; 98 °C 10s, 50 °C 15s, 60 °C 2min 40s, 33 cycles; 60 °C 7min; 16 °C storage; 1% agarose gel electrophoresis recovery.
  • the primer sequence is:
  • Step 2 Amplify the coding frames of GFP and renilla luciferase, and use pARM-GFP/RUCki constructed by Lu as the template (for specific construction methods, see Lu, J., et al. (2016). "Aredesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.”Parasit Vectors 9:198.), add the BtgZ I site at the 5'end of the GFP coding region, and add another BtgZ I site at the 3'end of the renilla luciferase coding region, you need to knock When importing other genes, cut this vector with BtgZI, the resulting framework will contain the complete promoter and terminator, and insert the gene to be expressed;
  • the reaction system for PCR amplification is the same as Table 1.
  • the reaction conditions are: 98°C 30s; 98°C 10s, 50°C 15s, 60°C 2min, 33 cycles; 60°C 7min; 16°C storage.
  • the primer sequence is:
  • reaction system is the same as Table 1, the reaction conditions are: 98 °C 30s; 98 °C 10s, 50 °C 15s, 60 °C 3min30s, 33 cycles; 60 °C 7min; stored at 16 °C, 1% agarose gel electrophoresis recovery 5'ef1a-gfp-ruc-3'pbdt fusion band, this band is the coding frame of GFP and renilla luciferase.
  • the rapid cloning method connects the AmpR-ori fragment to the GFP and renilla luciferase coding frames.
  • the amount of reaction system, plasmid frame and insert used is calculated according to the kit instructions.
  • the plasmid was directly sequenced and identified, and the analysis of sequencing results was analyzed with Snapgene software.
  • target sequence ie sgRNA sequence should be 5'-N(19)GG, 5'-N( 20) GG or 5'-N (21) GG sequence, 5'-N (20) GG sequence is preferred; since this application does not use in vitro transcription, but only constructs a common plasmid vector, the sgRNA sequence of this application refers to sgRNA Corresponding DNA sequence.
  • Step 1 Synthesize the pf47sgRNA sequence and anneal the primers.
  • the specific operations are as follows:
  • the primer was diluted with water to a concentration of 10uM, and annealed on the PCR instrument using the reaction system and procedures shown in Table 3:
  • Annealing reaction program 98°C 30s; 45°C 10min; 4°C storage;
  • the second step the pCBS-yfcu plasmid was cut with BtgZI, the reaction system and procedures were operated according to the BtgZI instruction manual, and the plasmid fragments were electrophoresed on 1.0% agarose gel.
  • the rapid cloning method connects the pf47sgRNA annealing product to the single-digested pCBS-yfcu plasmid.
  • the amount of the reaction system, plasmid fragments, and annealing product is calculated according to the kit instructions.
  • the transformation operation and subsequent single colony picking, expansion culture, strain preservation, and plasmid extraction are as described above.
  • the plasmid was directly sequenced and identified.
  • the sequence of the sequencing primer was SEQ ID NO. 19: GTATGTACACAATTATTTATAAGTCCAA; the analysis of the sequencing results was analyzed with Snapgene software.
  • pCBS-yfcu-pf47 plasmids are prepared in large quantities using the Magen Plasmid Extraction Kit. Since one electroporation experiment requires about 200 ⁇ g-400 ⁇ g plasmids and the plasmid concentration needs to be greater than 2 ⁇ g/ ⁇ L, the following procedures are used to concentrate the plasmids:
  • the first step use XhoI to perform single digestion of the pARM-BtgZI (GFP/RUC) BtgZI vector.
  • the digestion reaction conditions were operated according to the XhoI instruction manual, and the vector fragments were recovered by 1% agarose gel electrophoresis.
  • Step 2 Amplify the 5'homology arm of pf47 gene using Pf3D7 worm blood as template, the PCR reaction system is the same as Table 1, the reaction procedure is: 98°C 30s; 98°C 10s, 50°C 15s, 60°C 40s, 33 cycles ; 60 °C 7min; 16 °C stored; 1% agarose gel electrophoresis recovery band;
  • the primer sequence is as follows:
  • sequence of 5'armF is SEQ ID NO.20:
  • sequence of 5'armR is SEQ ID NO.21:
  • third step Connect the 5'homology arm of the pf47 gene to the pARM-BtgZI (GFP/RUC) BtgZI vector after single digestion with XhoI by rapid cloning, ligation, transformation, single colony picking, expansion, culture preservation, plasmid extraction as before As mentioned, the plasmid was sent for sequencing to confirm that the 5'homology arm was inserted correctly; the vector constructed in this step was pARM-BtgZI (GFP/RUC) BtgZI-pf475'arm;
  • the first step the above pARM-BtgZI (GFP/RUC) BtgZI-pf475'arm vector was single digested with KpnI, and the enzyme digestion reaction conditions were operated according to the KpnI instruction manual. Vector fragments were recovered by 1% agarose gel electrophoresis.
  • Step 2 Amplify the 3'homology arm of the pf47 gene using Pf3D7 worm blood as the template.
  • the PCR reaction system is the same as Table 1.
  • the reaction conditions are: 98°C30s; 98°C10s, 50°C15s, 60°C50s, 33 cycles; 60°C for 7min; 16°C for storage; 1% agarose gel electrophoresis to recover the band;
  • the primer sequence is as follows:
  • sequence of 3'armF is SEQ ID NO.22:
  • sequence of 3'armR is SEQ ID NO.23:
  • the rapid cloning method connects the 3'homology arm of the pf47 gene to the pARM-BtgZI (GFP/RUC) BtgZI-pf475'arm vector after single digestion with KpnI, ligation, transformation, single colony picking, expansion, culture preservation,
  • the plasmid extraction is the same as before; send the plasmid to sequencing to confirm that the 3'homology arm is inserted correctly.
  • the vector constructed in this step is pARM-BtgZI (GFP/RUC) BtgZI-pf47. This vector will be used as a general vector for the next step of plasmid construction. It can also be used as a positive control plasmid for electroporation.
  • Step 1 Insert the artificial PfEMP1R787OD recombinant protein with a length of 4616bp into the pf47 gene locus of pf3D7, construct the artificial PfEMP1R29var-V5-GFP-TM-ATS of the R787OD recombinant protein, and add "Age I" to the N-terminal domain of its R29var protein "Restriction site-GGPSG linking peptide-PstIrestriction site", SacIrestriction site was added after V5 tag, "SacIrestriction site-GGPSG connection peptide-NheIrestriction site” was added after GFP, encoding
  • the box is 4614 bp long.
  • the recombinant protein is driven by PfEF1 ⁇ 5'UTR and terminated by PbDT 3'UTR.
  • the size of the entire expression box is 6286 bp.
  • Step 2 Construct pARM-R787OD-Pf47 vector without GFP reporter gene.
  • R29var-V5-GFP-TM-ATS add "Age I restriction site-GGPSG linking peptide-Pst I restriction site” after the N-terminal domain of R29var protein, and add "Sac I restriction site-GGPSG” after the V5 tag Connect the peptide-NheI restriction site” to obtain the R787DGOI fragment, and then pass
  • the rapid cloning method was connected to the universal framework of the pARM-BtgZI (GFP/RUC) BtgZI-Pf47 plasmid.
  • the R787OD fragment was directly synthesized by Universal Biosystems (Anhui) Co., Ltd. and ligated into the pUC57 vector (pUC57-R29-TmATS).
  • the PCR reaction system of the R787OD fragment is the same as Table 1, the reaction procedure: 98 °C 30s; 98 °C 10s, 50 °C 15s, 60 °C 5min, 32 cycles; 60 °C 7min; stored at 4 °C, 1% agarose gel electrophoresis to recover R787OD Fragment.
  • PCR primer sequences are as follows:
  • sequence of 160817Fr29TmATS is SEQ ID NO.24:
  • sequence of 160817Rr29TmATS is SEQ ID NO.25:
  • Plasmid framework preparation BtgZI single digestion of pARM-BtgZI (GFP/RUC) BtgZI-Pf47 plasmid to recover large fragments, the digestion system and reaction procedure are the same as described above, and the large fragments are recovered by 1% agarose gel after digestion .
  • the R787DGOI fragment was ligated with the pARM-BtgZI (GFP/RUC) BtgZI-pf47 vector after single digestion with BtgZI by rapid cloning, ligation, transformation, single colony picking, expansion, culture preservation, and plasmid extraction as described above.
  • the plasmids were digested and identified by SacI, SalI and XbaI, and detected by 1% agarose gel electrophoresis.
  • the plasmids with positive digestion identification were sent for sequencing.
  • the sequencing primers are as follows:
  • sequence of 16930FseqR787 is SEQ ID NO. 26: ACACATTAAGTGGTAACAAACA
  • sequence of 16930RseqR787-1 is SEQ ID NO.27: CAGATCCATCATCATGTGCG
  • sequence of 16930RseqR787-2 is SEQ ID NO. 28: CGTAACAGGTGTGCATTTC
  • sequence of 16930RseqR787-3 is SEQ ID NO. 29: ATGCTTATCCTCATCGTGGTG
  • sequence of 16930RseqR787-4 is SEQ ID NO. 30: TCTTCGTCTGTATCTCCTTCAAC
  • sequence of 16930RseqR787-5 is SEQ ID NO.31: CTAAACGTTCATGATGATTTTCCC
  • Step 3 Construct the pARM-R787OD-Pf47 vector; insert the GFP gene at the Sac I site of pARM-R787OD-Pf47 and use Rapid cloning ligation, amplification of ORF fragments of GFP: Lu et al. (Lu, J., et al. (2016). "A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum.” Parasit Vectors 9: 198.)
  • the constructed pARM-GFP/RUCki plasmid is used as the template.
  • the PCR system is the same as Table 1.
  • the reaction procedure is as follows: 30s; 98°C 10s, 50°C 15s, 60°C 1min 30s, 33 cycles; 60°C 7min; 16°C storage , 1% agarose gel electrophoresis recovery.
  • the R787OD fragment was ligated with the pARM-R787OD-Pf47 vector digested with Sac I by rapid cloning, incubated at 37°C for 3h, and recovered by 1% agarose gel electrophoresis.
  • the transformation operation and subsequent single colony picking, expansion culture, strain preservation, and plasmid extraction are as described above.
  • the plasmid was digested with SacI and identified.
  • the plasmids identified by enzyme digestion were sent for sequencing.
  • the sequencing segment was ORF of GFP.
  • the sequencing primers were as follows:
  • sequence of 161024FseqGFP is SEQ ID NO.32:
  • sequence of 161024RseqGFP is SEQ ID NO.33:
  • the software used for the analysis of sequencing results, the mass preparation and concentration of plasmids are as described above.
  • P. falciparum 3D7 human P. falciparum strains were cultured in vitro using standardized conditions: including the use of complete medium (10.4g/L RPMI 1640, 25mM HEPES, 0.5% (wt/vol) AlbuMAX I, 1.77mM sodium bicarbonate, 100 ⁇ M hypoxanthine, 12.5 ⁇ g/mL gentamicin sulfate, pH 7.2), 1% hematocrit, 37°C three-gas incubator (1% O 2 , 3% CO 2 , and 96% N 2 ) to cultivate.
  • complete medium (10.4g/L RPMI 1640, 25mM HEPES, 0.5% (wt/vol) AlbuMAX I, 1.77mM sodium bicarbonate, 100 ⁇ M hypoxanthine, 12.5 ⁇ g/mL gentamicin sulfate, pH 7.2
  • 1% hematocrit 37°C three-gas incubator (1% O 2 , 3% CO 2 , and 9
  • Giemsa staining the number of infected red blood cells / total red blood cells, generally count the number of cells around 10 fields of view, the total number of red blood cells ⁇ 1000; decide whether to change the fluid according to the level of infection rate When the infection rate is higher than 5%, change the fluid once a day, and the interval between fluid changes should not exceed 24 hours. When the infection rate of Plasmodium is about 5%, the schizonts can be obtained by gelatin enrichment.
  • the infection rate reaches 80-90% (try to use the worm blood with a high initial infection rate to achieve this infection rate within 48h), start adding Blastidicin S to a final concentration of 5 ⁇ g/mL for drug sieve culture, and add 100 ⁇ l 50 %RBC, next, every 24h smear examination, generally the growth of insects is basically controlled after 24h of drug addition, the infection rate no longer rises, basically no live insects are seen in about 4 days, and the drug pressure starts to be withdrawn in about 10 days Medicine culture. If the insertion of the exogenous fragment is successful, live insects can be seen again after 20-30 days; when the drug is being screened, 50 ⁇ L of 50% hematocrit is supplemented weekly.
  • the PCR method was used to detect the integration of the R787OD insert in the malaria parasite genome at the DNA level.
  • the PCR template was worm blood.
  • the primer sequence and PCR reaction conditions were as follows:
  • the reaction system is shown in Table 1 (the system can be reduced to 25 ⁇ L, and the volume of all components added is reduced proportionally).
  • the reaction procedure is: 98°C 30s; 98°C 10s, 50°C 15s, 60°C 9min, 33 cycles; 60°C 9min; 16°C storage; the amplified product (8.0kb) of the R787OD expression box is sequenced and identified by sequencing The company designed primer sequencing by itself;
  • the R787OD expression box contains GFP, theoretically, the expression of GFP can be observed with a fluorescence microscope, however, the observation results of the fluorescence microscope show that the resulting transgenic Plasmodium has no green fluorescence, the reason may be that GFP is located in the 787 amino acid R787 domain and 478 The amino acid "transmembrane region-ATS intracellular region" may encounter greater steric hindrance and cannot be folded correctly.
  • RNAiso Blood (Takara, Code No. 9113) kit to extract wild-type Pf3D7 and R787OD transgenic Plasmodium RNA, respectively, The specific operation is carried out according to the instructions of the kit; for reverse transcription II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R223-01), the specific operation is carried out according to the kit instructions; because the R787OD fusion gene contains the GFP reporter gene, GFP can be used as the target gene for RT-PCR detection.
  • the primers are as follows:
  • the amplification length of this pair of primers is 263bp, and there are also detection primers for the internal reference gene actin:
  • the amplification length is 287bp
  • RT-PCR detection reaction system is the same as Table 1.
  • the results show that the target gene can be transcribed normally, as shown in Figure 6(A) and Figure 6(B); at the same time, Western blot is also used to detect the expression of the target gene at the protein level.
  • the Western blot test method is as follows:
  • Tris-HCl (pH6.8): Take 12.14g Tris-base, add 60mL of deionized water to fully dissolve, adjust the pH to 6.8 with concentrated hydrochloric acid, deionized water to a constant volume of 100mL, store at room temperature for later use;
  • Tris-HCl (pH8.8): Take 27.23g Tris-base, add 80mL of deionized water to fully dissolve, adjust the pH value to 8.8 with concentrated hydrochloric acid, make up to 150mL with deionized water, store at room temperature for later use;
  • 5 ⁇ Tris-glycine electrophoresis buffer Weigh 15g Tris-base, 94g glycine, 5g SDS, add 800mL of deionized water, stir to dissolve, make up to 1L with deionized water, store at room temperature for later use;
  • 5 ⁇ membrane transfer buffer Weigh 15g Tris-base, 72g glycine, add 800mL deionized water to dissolve, make up to 1L with deionized water, store at room temperature for future use, if using PVDF membrane or NC membrane, add 20% methanol;
  • TBS buffer solution Weigh 8.8g of sodium chloride, add 20mL of 1M Tris-HCl (pH8.0), add 800mL of deionized water, fully dissolve and make up to 1L with deionized water, store at room temperature for future use (refer to "TaKaRa commodity The preparation method of the appendix of the catalogue 2012-2013);
  • TBST buffer Take 0.5mL of Tween-20, add TBS and stir until it is fully dissolved and dilute to 1L, and store at room temperature until use;
  • SDS-PAGE glue Refer to the SDS-PAGE glue preparation method in the appendix of "TaKaRa Catalog 2012-2013" for preparation, or use pre-made glue directly.
  • Electrophoresis load 20 ⁇ L, use 80V for concentrated gel and run for 30min; use 120V for separating gel;
  • PVDF membrane is activated before use (soaked in methanol for 10s, and then immediately immersed in transfer membrane buffer), the transfer membrane is 100V, 1.5h, and the electrophoresis tank is placed in an ice water bath;
  • Blocking After the film transfer is successful, wash with TBST, then soak the film in 5% skimmed milk powder/TBST, and seal at 4°C overnight;
  • Secondary antibody incubation After the primary antibody is incubated, wash the membrane with TBST, and then incubate the secondary antibody.
  • the secondary antibody (Goat Anti-Mouse IgG H&L, Abcam, ab205719) is diluted in 5% skim milk powder at a ratio of 1:5,000. In TBST, incubate at room temperature for 1h;
  • the expression of the target gene in red blood cells was further detected by immunofluorescence method.
  • the immunofluorescence detection method is as follows:
  • PBSTx Add Triton X-100 to PBS to a final concentration of 0.1%, mix well, that is PBSTx;
  • Blocking solution 5% BSA/PBSTx 1g BSA dissolved in 20mL PBS, divided into 1mL/tube and stored at -20°C, take an appropriate amount to thaw before the experiment, add Triton X-100 to a final concentration of 0.1%;
  • Anti-GFP Rabit pAb working solution 1:250 dilution, that is, adding 250 ⁇ L of blocking solution 5% BSA/PBSTx per 1 ⁇ L of Anti-V5 mouse mAb;
  • Goat Anti-Rabbit IgG H&L 1:500 dilution, that is, add 500 ⁇ L of blocking solution 5% BSA/PBSTx for every 1 ⁇ LCy3 Goat anti-mouse IgG;
  • Anti-V5 mouse mAb working solution 1:250 dilution, that is, every 1 ⁇ LL Anti-V5 mouse mAb is added 250 ⁇ L of blocking solution 5% BSA/PBSTx;
  • Cy3 Goat anti-mouse IgG working solution 1:500 dilution, that is, add 500 ⁇ L of blocking solution 5% BSA/PBSTx for every 1 ⁇ LCy3 Goat anti-mouse IgG.
  • DAPI (5ug/mL) working solution DAPI stock solution (5mg/mL) was diluted 1:1000, that is, 500 ⁇ L of blocking solution 5% BSA/PBSTx was added per 1 ⁇ LDAPI working solution.
  • Protozoa rate reached more than 5% add 5-FC to a final concentration of 38 ⁇ M for negative screening, change the liquid every day until no live insects were observed in the smear, change the liquid every other day, negative sieve lasts for 2-3 weeks, smear You can see live insects.
  • the infection rate when the infection rate reaches 3% or more, it will be monoclonalized by the limiting dilution method, and the monoclonal will be obtained in about 3 weeks.
  • Intracellular immunofluorescence detection showed that the R787OD monoclonal worm strain obtained by negative screening, the target protein was distributed in the cytoplasm of erythrocytes, which was consistent with the malaria parasite that was not negatively screened (see Figure 9); The expression of foreign genes in transgenic Plasmodium has an impact.
  • the "Microplate-SYBR Green I” method was used to detect the sensitivity of different R787OD monoclonals to Blasticidin S. The results showed that the IC50 value was between 0.89-0.94 ⁇ g/mL, while the wild-type Plasmodium was 1.02 ⁇ g/mL.
  • the target with the lowest possibility of off-target in the genome is selected. Its sequence is TATAATCACCCCTGGATTCTGG. pCBS-yfcu-PfNT1 vector;
  • the GFP tag sequence knocked in is SEQ ID NO. 44:
  • the 3’ homology arm sequence is SEQ ID NO.45:
  • PCR was used to detect the integration of the GFP tag at the N-terminus of the PfNT1 gene.
  • the PCR primer sequences are shown in Table 5; PCR and sequencing results indicate that the GFP tag has been accurately inserted into the PfNT1 gene.
  • the expression of GFP can be observed with a fluorescence microscope.
  • the gene editing system provided in this application is based on the endogenous U6 promoter-dependent vector system, a negative selection marker is added to the Cas9 expression cassette of the expression vector, and the anti-resistance vector on the rescue vector containing the homology arm is added.
  • the drug screening marker is transferred to the expression vector, reducing the basic size of the rescue vector and expanding its capacity, which can mediate the knock-in of 6.3 kb foreign gene fragments in human Plasmodium, and the obtained recombinant Plasmodium does not contain drug-resistant screening
  • the marker and the residue of the Cas9 protein expression plasmid can be used for continuous gene editing using the same drug sieve marker. The performance is stable, efficient and concise, powerful, and has broad application prospects and huge market value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système d'édition de gènes basé sur la technologie CRISPR-Cas9 et son utilisation. Le système d'édition de gènes est basé sur un système de vecteur dépendant du promoteur U6 endogène, Un marqueur de sélection négative est ajouté dans une cassette d'expression de Cas9 d'un vecteur d'expression, et des marqueurs de sélection résistant aux médicaments sur des vecteurs de sauvetage comprenant des bras homologues sont transférés sur le vecteur d'expression. Par conséquent, la taille de base des vecteurs de sauvetage est réduite et la capacité est augmentée, le knock-in d'un segment de gène exogène long de 6.3 kb dans le plasmodium peut être médié, et le plasmodium recombiné obtenu n'a pas de marqueurs de sélection résistant aux médicaments et de résidus de plasmide d'expression de protéine Cas9.
PCT/CN2019/082198 2019-01-11 2019-04-11 Système d'édition de gènes basé sur la technologie crispr-cas9 et son utilisation WO2020143125A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910026600.8A CN111434772A (zh) 2019-01-11 2019-01-11 一种基于CRISPR-Cas9技术的基因编辑系统及应用
CN201910026600.8 2019-01-11

Publications (1)

Publication Number Publication Date
WO2020143125A1 true WO2020143125A1 (fr) 2020-07-16

Family

ID=71521404

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/082198 WO2020143125A1 (fr) 2019-01-11 2019-04-11 Système d'édition de gènes basé sur la technologie crispr-cas9 et son utilisation

Country Status (2)

Country Link
CN (1) CN111434772A (fr)
WO (1) WO2020143125A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019051642A1 (fr) * 2017-09-12 2019-03-21 广州中科蓝华生物科技有限公司 Kit pour la transfection de parasites intracellulaires et son utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108486145A (zh) * 2018-02-12 2018-09-04 中国科学院遗传与发育生物学研究所 基于CRISPR/Cas9的植物高效同源重组方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103898140B (zh) * 2014-04-14 2017-03-22 中国科学院天津工业生物技术研究所 一种简便高效的基因编辑方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108486145A (zh) * 2018-02-12 2018-09-04 中国科学院遗传与发育生物学研究所 基于CRISPR/Cas9的植物高效同源重组方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN, JIANWU ET AL.: "A Double Fluorescence Screening Strategy to Enhance the Efficiency of Gene Targeting", CHINA BIOTECHNOLOGY, vol. 37, no. 1, 31 December 2017 (2017-12-31), pages 58 - 63, ISSN: 1671-8135 *
FRANFOIS WASELS: "A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum", JOURNAL OF MICROBIOLOGICAL METHODS, 10 June 2017 (2017-06-10), XP055526146, ISSN: 0167-7012 *
INGA SOREANU: "Marker-free genetic manipulations in yeast using CRISPR/ CAS9 system", CURRENT GENETICS, 6 April 2018 (2018-04-06), XP036591507, ISSN: 0172-8083, DOI: 10.1007/s00294-018-0831-y *
JUNNAN LU: "A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum", PARASITES & VECTORS, 31 December 2016 (2016-12-31), XP055724875, ISSN: 1756-3305 *

Also Published As

Publication number Publication date
CN111434772A (zh) 2020-07-21

Similar Documents

Publication Publication Date Title
US11555181B2 (en) Engineered cascade components and cascade complexes
US20220315892A1 (en) Engineered Artificial Antigen Presenting Cells for Tumor Infiltrating Lymphocyte Expansion
AU2019240724B2 (en) Engineered nucleic-acid targeting nucleic acids
JP6715419B2 (ja) カンピロバクター・ジェジュニｃｒｉｓｐｒ／ｃａｓシステムに由来するｒｇｅｎを使用したゲノム編集
JP2023071855A (ja) ＣＲＩＳＰＲ－Ｃａｓエフェクターポリペプチド及びその使用方法
JP2023030067A (ja) Ｒｎａ誘導型核酸修飾酵素及びその使用方法
WO2020236972A2 (fr) Systèmes de ciblage d'acides nucléiques à constituants multiples autres que de classe i
JP2009165497A (ja) ｍＲＮＡの阻害／不安定領域の除去方法
CN111575319B (zh) 一种高效的crispr rnp和供体dna共位介导的基因插入或替换方法及其应用
CN109153990A (zh) 用于基因编辑的组合物和方法
JP2020528760A (ja) 新規融合タンパク質の調製およびそのタンパク質合成の向上における使用
CN114981423A (zh) I-b型crispr相关转座酶系统
BR112014002663A2 (pt) método para fornecer um ácido nucleico para uma organela não nuclear em uma célula, método para produzir uma célula vegetal geneticamente modificada, célula vegetal geneticamente modificada, método para produzir uma planta geneticamente modificada, planta geneticamente modificada, método para produzir uma célula animal geneticamente modificada, nanocarreador direcionado a mitocôndria e nanocarreador direcionado a cloroplasto
CN111040998B (zh) 一种稳定表达狂犬病毒糖蛋白细胞株的构建及其应用
WO2020143125A1 (fr) Système d'édition de gènes basé sur la technologie crispr-cas9 et son utilisation
CN103732751A (zh) 在幽门螺杆菌中的基因表达和根除系统
US20020055172A1 (en) Multiple promoter expression constructs and methods of use
US20160186196A1 (en) Method and composition for generating programmed cell death resistant algal cells
Upadhya et al. Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii
WO2023216415A1 (fr) Système d'édition de bases fondé sur la complémentation bimoléculaire de la désaminase, et son utilisation
US20150344895A1 (en) Methods of modifying algal cell genomes
WO2001098453A2 (fr) Vecteurs d'expression comprenant un fragment modifie de l'operon tryptophane
CN105368865B (zh) 可控的基因组修饰疟原虫、重组表达载体及构建方法、应用
EP0140762A1 (fr) Vecteurs d'expression d'une proteine antigenique de la rage dans les cellules eucaryotes et leur application à la préparation d'un vaccin
CN113528514B (zh) 基于CRISPR-Cas12a干预阻断病毒逆转录转座的技术

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19909047

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 30/11/2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19909047

Country of ref document: EP

Kind code of ref document: A1