Microsporidia gene deletion strain of cryptosporidium parvum with deletion of dg9 and / or dg10 genes and application thereof

Abstract

The application discloses a microsporum gene deletion strain of Cryptosporidium parvum with deleted DG9 and / or DG10 genes and application thereof. The microsporum gene deletion strain of Cryptosporidium parvum is obtained by directly knocking out dense granule protein DG9 genes and / or DG10 genes of Cryptosporidium parvum through a gene editing technology. The application discloses the regulation of Cryptosporidium parvum dense granule proteins DG9 and DG10 on the pathogenicity of Cryptosporidium parvum. Research shows that the strain with deleted DG9 or DG10 genes or double gene deletion can reduce the clinical symptoms of infected mice and prolong the survival time of the mice. The virulence of the gene deletion strain is weakened, the worm load in the mice is reduced, and the excretion intensity of oocysts is weakened, so that the gene deletion strain has the advantage of weakened virulence and can be used for developing more attenuated vaccines and therapeutic drugs of Cryptosporidium parvum, thereby laying a solid foundation for the prevention and control of Cryptosporidium parvum.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedicine, and more specifically, to a Cryptosporidium microsporidium gene-deleted strain lacking the DG9 and / or DG10 genes and its applications. Background Technology

[0002] Cryptosporidium microsporum ( Cryptosporidium parvum Cryptosporidium is an important apical protozoan, widely distributed in animals, humans, and the environment. It is primarily transmitted via the fecal-oral route, parasitizing the intestinal mucosal epithelial cells of animals and humans. Infection leads to cryptosporidiosis, characterized by digestive disorders and watery diarrhea, and in severe cases, death. Cryptosporidium infection rates are high, exceeding 70% in young cattle and sheep. Furthermore, its ability to spread across species and infect humans poses a serious threat to both the livestock industry and public health.

[0003] Currently, prevention and treatment options for cryptosporidiosis are very limited. Only nitrozonide has been approved by the FDA for clinical treatment of cryptosporidiosis in humans. Nitrozonide is effective in treating cryptosporidiosis in immunocompetent adults and children over one year of age; however, it is ineffective in patients with HIV and children with underdeveloped immune systems. Furthermore, there is currently no effective vaccine for the prevention of cryptosporidiosis. Existing peptide and subunit vaccines only produce very low partial immunoprotective effects and are still in the research stage; there are currently no commercially available and effective cryptosporidiosis vaccines on the market. Simultaneously, because Cryptosporidium is difficult to passage continuously in vitro and undergo reverse genetic manipulation, its genetic manipulation technology has long been underdeveloped, resulting in insufficient understanding of the invasion and development mechanisms of Cryptosporidium. Therefore, no relevant vaccine candidate molecules have been researched and identified.

[0004] Dense granular proteins play a crucial role in apical-complexal parasites and may be potential targets for combating Cryptosporidium infection. However, few functional relationships of dense granular proteins have been publicly disclosed in Cryptosporidium parvum. Existing research has shown that the dense granular protein gene cgd7_4500 (DG6) is associated with virulence, but no relationship has been found between other dense granular proteins and their related genes and the pathogenicity and virulence of Cryptosporidium. Since there is currently a lack of vaccines or gene-deleted strains for the prevention and control of Cryptosporidium, it is necessary to develop more Cryptosporidium vaccine strains or products for better prevention and control of cryptosporidiosis. Therefore, this invention application is filed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing Cryptosporidium vaccine strains. The present invention provides a Cryptosporidium microsporidium gene deletion strain that lacks the DG9 and / or DG10 genes and its application.

[0006] The first objective of this invention is to provide a Cryptosporidium microsporidium gene-deleted strain.

[0007] The second objective of this invention is to provide a method for constructing a Cryptosporidium microsporidium gene-deleted strain.

[0008] A third objective of this invention is to provide the application of Cryptosporidium microsporidium gene-deleted strains.

[0009] A fourth objective of this invention is to provide the application of reagents that delete or knock out the Cryptosporidium microsporidium DG9 and / or DG10 genes.

[0010] The fifth objective of this invention is to provide the application of Cryptosporidium microsporidium DG9 and / or DG10 genes as targets.

[0011] The sixth objective of this invention is to provide a Cryptosporidium microsporidium vaccine.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention provides a Cryptosporidium microsporidium gene deletion strain, which is obtained by directly knocking out the DG9 gene and / or the DG10 gene of Cryptosporidium microsporidium dense granule protein using gene editing technology; the nucleotide sequence of the DG9 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the DG10 gene is shown in SEQ ID NO: 2.

[0014] This invention, through spatial proteomics and comparative genomics analysis, discovered that the cgd8_680_690 gene (named the DG10 gene) is only present in... C. parvum It exists in, and in C. hominis The deletion of this gene may involve its role in host adaptive regulation. This gene shares high similarity (71.8% and 57.3%) with the adjacent cgd8_660_670 gene (named DG9) at the 5' and 3' ends, suggesting they may belong to the same functional family, and that sequence polymorphism may affect virulence differences. Further CRISPR / Cas9 gene editing was used to compare the in vitro growth and in vivo infection phenotypes of the gene-deleted strain and the wild-type strain, revealing their roles in Cryptosporidium growth, development, and pathogenicity. The results showed that neither dg9 nor dg10 are essential genes for Cryptosporidium growth and development, but they are related to the pathogenicity of Cryptosporidium. This invention will deepen our understanding of the function of dense granular proteins and provide new strategies for the prevention and control of cryptosporidiosis.

[0015] This invention uses CRISPR / Cas9 gene editing technology to directly knock out the dg9 and dg10 genes, obtaining... D dg9 , Δdg10 Single-deletion strains and Δdg9Δdg10 In vitro and in vivo infection experiments with the double-deleted strains showed that, in HCT-8 cell culture, deletion of either DG9 or DG10 alone could alleviate clinical symptoms and prolong mortality in infected mice; simultaneously, double deletion of DG9 or DG10 also alleviated clinical symptoms and prolonged survival time in infected mice. The gene-deleted strains constructed in this invention all exhibit reduced virulence, with lower parasite load and weaker oocyst excretion in mice. This facilitates the development of new drug targets or the preparation of gene knockout strains as candidate attenuated vaccines, laying a solid foundation for the control of Cryptosporidium microsporidium and providing more attenuated vaccine strains.

[0016] This invention provides a method for constructing a Cryptosporidium microsporidium gene-deleted strain, comprising the following steps: S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as a template, construct double sgRNA CRISPR plasmids pCRISPR-U6-Δdg9 or pCRISPR-U6-Δdg10 using DG9 or DG10 gene sgRNA primers. S2. Using pIb-tdTomato-Nluc-P2A-neo plasmid and wild-type Cryptosporidium microsporum DNA as templates, construct homologous recombinant plasmids pd-Δdg9-tdTomato-Nluc-P2A-neo or pd-Δdg10-tdTomato-Nluc-P2A-neo plasmids using DG9 or DG10 gene primers; S3. The double sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into wild-type Cryptosporidium microsporidium strains. Through drug screening and PCR identification, Cryptosporidium microsporidium strains with DG9 or DG10 gene deletions were obtained. Δdg9 or Δdg10 ; or S11. Using pCRISPR-U6- Δdg9 The pCRISPR-U6-gRNA2 plasmid, pCRISPR-U6-gRNA1 plasmid, and the homologous recombinant plasmid pCRISPR-U6-gRNA1 plasmid were constructed using the whole genome of Cryptosporidium microsporum as a template and DG9DG10 gene primers. D dg9Δdg10 ; S21. Using pIb-tdTomato-Nluc-P2A-neo plasmid and wild-type Cryptosporidium microsporum DNA as templates, and employing DG9DG10 gene primers, construct the homologous recombinant plasmid pd- Δdg9Δdg10 -tdTomato-Nluc-P2A-neo; S31. The double sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into wild-type Cryptosporidium microsporidium strains. The DG9DG10 double gene deletion strain of Cryptosporidium microsporidium was obtained through drug screening and PCR identification. Δdg9Δdg10 .

[0017] Preferably, in step S1, using pCRISPR-U6-DG9-6HA and pCRISPR-U6-Δdg9-g2 plasmids as templates and DG9 gene sgRNA primers, a double sgRNA CRISPR plasmid pCRISPR-U6-Δdg9 is constructed; using pCRISPR-U6-DG10-smHA and pCRISPR-U6-Δdg10-gRNA2 plasmids as templates and DG10 gene sgRNA primers, a double sgRNA CRISPR plasmid pCRISPR-U6-Δdg10 is constructed.

[0018] Preferably, the primer sequences for the DG9 or DG10 gene sgRNA in step S1 are as shown in SEQ ID NO: 3~8.

[0019] Preferably, the DG9 or DG10 gene primers in step S2 and the DG9 / DG10 gene primers in step S21 are as shown in SEQ ID NO: 9-20, respectively.

[0020] Preferably, the wild-type Cryptosporidium microsporidium strain is the Hebei IIdA20G1 subtype of wild-type Cryptosporidium microsporidium.

[0021] As a preferred embodiment, the present invention provides a more specific method for constructing Cryptosporidium microsporidium DG9 gene-deleted strains, DG10 gene-deleted strains, and DG9 / DG10 double gene-deleted strains: (1) The starting strain was the wild-type Cryptosporidium microsporum Hebei IIdA20G1 subtype strain of Cryptosporidium genus of Cryptosporidium family of order Tuccinidae with DG9 and DG10 genes. (2) Construction of the double sgRNA CRISPR plasmid pCRISPR-U6-Δdg9: Using pACT1::Cas9-GFP-U6::sgTK plasmid as a template, the TK target-specific sgRNA was amplified using upstream and downstream primers. The sgRNA was replaced with the target-specific sgRNA1 of the DG9 gene or DG10 gene. The plasmid was ligated using the principle of homologous recombination and then transformed into DH5α to obtain the pCRISPR-U6-DG9-6HA or pCRISPR-U6-DG10-6HA plasmids. The pCRISPR-U6-Δdg9-g2 or pCRISPR-U6-Δdg10-g2 plasmids were constructed using the same method. Then, using pCRISPR-U6-DG9-6HA or pCRISPR-U6-DG10-6HA plasmids as templates, the plasmid backbone was amplified with primers; subsequently, using pCRISPR-U6-Δdg9-g2 or pCRISPR-U6-Δdg10-g2 plasmids as templates, the sgRNA fragment was amplified, and finally the plasmids pCRISPR-U6-Δdg9 or pCRISPR-U6-Δdg10 were constructed. (3) Construction of homologous recombination donor plasmid: It is obtained by linking the 5' and 3' homologous arms of the DG9 or DG10 gene to express the spontaneous red fluorescent protein Tdtomato sequence, the drug screening resistance gene P2A-neomycin, and the luciferase value determination gene Nluc. Specifically: using the genome of the starting strain *Cryptospora microsporum*, Hebei subtype IIdA20G1, as a template, primers were designed to amplify the -5'UTR and -3'UTR of the DG9 or DG10 gene; using the pIb-tdTomato-Nluc-P2A-neo plasmid as a template, primers were designed to amplify the tdTomato-Nluc-P2A-neo fragment; using the p5520-5510-2-6HA-Nluc-P2A-neo plasmid as a template, primers were designed to amplify the ampicillin-resistant plasmid backbone fragment; finally, the four DNA fragments were ligated using the Uniclone one-step seamless cloning kit to construct recombinant plasmids pd-Δdg9-tdTomato-Nluc-P2A-neo or pd-Δdg10-tdTomato-Nluc-P2A-neo; (4) Cryptosporidium microsporidium gene deletion strain Δdg9 or Δdg10Obtaining: The sporozoite suspension obtained after decapsulation of the oocysts in step (1) was mixed with the pCRISPR-U6-Δdg9 or pCRISPR-U6-Δdg10 plasmid obtained in step (2) and the pd-Δdg9-tdTomato-Nluc-P2A-neo or pd-Δdg10-tdTomato-Nluc-P2A-neo plasmid obtained in step (3), and then electrotransfected into wild-type Cryptosporidium microsporidium strains to obtain Cryptosporidium microsporidium gene deletion strains Δdg9 or Δdg10.

[0022] Meanwhile, the Cryptosporidium microsporidium DG9DG10 double gene deletion strain Δdg9Δdg10 The construction method is as follows: the starting strain is the wild-type Cryptosporidium microsporum Hebei IIdA20G1 subtype of Cryptosporidium genus of Cryptosporidium family of order Trunciformes with DG9 and DG10 genes. Double sgRNA CRISPR plasmid pCRISPR-U6- Δdg9Δdg10 Construction: Using pCRISPR-U6-Δdg9-gRNA2 plasmid as a template, the plasmid backbone was amplified with primers; then, using pCRISPR-U6-DG10-smHA-gRNA1 plasmid as a template, the sgRNA fragment was amplified, and finally the plasmid pCRISPR-U6-Δdg9Δdg10 was constructed. Construction of the pd-Δdg9Δdg10-tdTomato-Nluc-P2A-neo homologous recombination donor plasmid: It was obtained by ligating the 5' homologous arm of the DG9 gene and the 3' homologous arm of the DG10 gene to express the spontaneous red fluorescent protein Tdtomato sequence, the drug screening resistance gene P2A-neomycin, and the luciferase value assay gene Nluc. Specifically: using the genome of the starting strain *Cryptospora microsporum* subtype IIdA20G1 from Hebei Province as a template, primers were designed to amplify the DG9-5'UTR and DG10-3'UTR; using the pIb-tdTomato-Nluc-P2A-neo plasmid as a template, primers were designed to amplify the tdTomato-Nluc-P2A-neo fragment; using the p5520-5510-2-6HA-Nluc-P2A-neo plasmid as a template, primers were designed to amplify the ampicillin-resistant plasmid backbone fragment; the four DNA fragments were ligated using the Uniclone one-step seamless cloning kit to construct the recombinant plasmid pd-Δdg9Δdg10-tdTomato-Nluc-P2A-neo; Obtaining the Cryptosporidium microsporidium double-deletion strain Δdg9Δdg10: The sporozoite suspension obtained after decapsulation of the oocysts was mixed with the pCRISPR-U6-Δdg9Δdg10 plasmid and the pd-Δdg9Δdg10-tdTomato-Nluc-P2A-neo plasmid obtained in the above steps, and then electrotransfected into wild-type Cryptosporidium microsporidium strain to obtain the Cryptosporidium microsporidium double-deletion strain Δdg9Δdg10.

[0023] This invention provides reagents for deleting or knocking out Cryptosporidium DG9 and / or DG10 genes for use in constructing DG9 and / or DG10 gene-deleted strains or in the preparation of Cryptosporidium vaccines.

[0024] This invention provides the application of reagents containing the deletion or knockout of the Cryptosporidium DG9 and / or DG10 genes in the preparation of Cryptosporidium attenuated products or in the in vitro inhibition of Cryptosporidium growth.

[0025] This invention provides the application of Cryptosporidium DG9 and / or DG10 genes as targets in the preparation of Cryptosporidium gene-deleted strains or in the preparation of Cryptosporidium vaccines.

[0026] This invention provides the application of Cryptosporidium DG9 and / or DG10 genes as targets in the preparation of products that reduce Cryptosporidium virulence or in the preparation of products that inhibit Cryptosporidium growth in vitro.

[0027] This invention provides the application of the above-mentioned Cryptosporidium microsporidium gene-deleted strain in the preparation of Cryptosporidium microsporidium vaccines.

[0028] The present invention also provides a Cryptosporidium microsporidium vaccine containing the above-mentioned Cryptosporidium microsporidium gene-deleted strain.

[0029] The present invention has the following beneficial effects: This invention is the first to discover that the dg9 and dg10 genes are not essential for the growth and development of Cryptosporidium parvum, but are related to its pathogenicity. Gene-deleted Cryptosporidium strains were successfully constructed using gene editing technology to delete the DG9 and / or DG10 genes. In vitro and in vivo infection experiments showed that deleting either DG9 or DG10 alone in HCT-8 cell culture reduced clinical symptoms and prolonged mortality in infected mice. Simultaneously, double deletion of DG9 and DG10 also reduced clinical symptoms and prolonged survival in infected mice. Furthermore, the genetic background of the double-deleted strains was more stable, with a significantly reduced risk of reversion mutations, resulting in higher reliability and controllability in subsequent attenuated vaccine applications. The gene-deleted strains constructed in this invention have the advantage of reduced virulence, lower parasite load in mice, and weaker oocyst excretion, effectively prolonging mouse survival. This provides researchers with insights into developing new drug targets or preparing gene knockout strains as candidate attenuated vaccines, laying a solid foundation for the control of Cryptosporidium parvum. Attached Figure Description

[0030] Figure 1 yes Δdg9、Δdg10、Δdg9Δdg10 Schematic diagram of the construction principle of the insect strain (A in the figure is...) Δdg9 Insect strain; B is Δdg10 Insect strain; C is Δdg9Δdg10 Insect strains).

[0031] Figure 2 yes Δdg9、Δdg10、Δdg9Δdg10 Agarose gel electrophoresis results of PCR identification of insect strains (A in the figure is...) Δdg9 Insect strain; B is Δdg10 Insect strain; C is Δdg9Δdg10 Insect strains).

[0032] Figure 3 It is to evaluate using in vitro culture experiments. Δdg9、Δdg10 Results of the in vitro growth of the insect strain (A in the figure is...) D dg9 Insect strain; B is Δdg10 Insect strains).

[0033] Figure 4 It assesses the amount of oocysts excreted from infected mice in vivo. Δdg10 Results of infection intensity of the parasite strain in mice.

[0034] Figure 5 It assesses the changes in body weight of infected mice in vivo. Δdg10、 Results of infection intensity of the parasite strain in mice.

[0035] Figure 6 It uses the survival rate of infected mice in vivo to assess Δdg10 Results of insect strain virulence.

[0036] Figure 7 It uses clinical symptoms of infected mice in vivo for assessment Δdg10 Results of insect strain virulence (A in the figure represents experimental mice; B represents statistical data).

[0037] Figure 8 It assesses the amount of oocysts excreted from infected mice in vivo. Δdg9、Δdg9Δdg10 Results of infection intensity of the parasite strain in mice.

[0038] Figure 9 It assesses the changes in body weight of infected mice in vivo. Δdg9、Δdg9Δdg10 Results of infection intensity of the parasite strain in mice.

[0039] Figure 10 It uses the survival rate of infected mice in vivo to assess Δdg9、Δdg9Δdg10 Results of insect strain virulence.

[0040] Figure 11 It uses clinical symptoms of mice infected in vivo for assessment Δdg9、Δdg9Δdg10 Results of insect strain virulence (A in the figure represents experimental mice; B represents statistical data). Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] The pACT1::Cas9-GFP-U6::sgTK plasmid and pINS1-3HA-Nluc-P2A-neo plasmid used in the examples were both from the laboratory of L. David Sibley at the University of Washington.

[0044] Typing identification of wild-type Cryptosporidium parvum subtype IIdA20G1 strains: Li N, Zhao W, Song S, Ye H, Chu W, Guo Y, et al. Diarrhea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairy calves. Transbound Emerg Dis. 2022;69:e1606-17.

[0045] Example 1 Cryptosporidium microphyllum Δdg9、Δdg10 Construction of single-knockout insect strains 1. Starting strain This embodiment, through previous spatial proteomics and comparative genomics analysis, discovered that the cgd8_680_690 gene (i.e., the DG10 gene) is only present in... C. parvum It exists in, and in C. hominis The deletion in the middle may be involved in host adaptive regulation. This gene has high similarity (71.8% and 57.3%) with the adjacent cgd8_660_670 (i.e., the DG9 gene) at the 5' and 3' ends, suggesting that they may belong to the same functional family, and that sequence polymorphism may affect virulence differences.

[0046] Therefore, the starting strain was the wild-type Cryptosporidium microsporum subtype Hebei IIdA20G1 strain (Cryptosporium microsporum isolate) belonging to the genus Cryptosporidium of the family Cryptosporidae in the order Clusteriae. C. parvum IIdA20G1-HE (this isolate was isolated and subcultured in our laboratory from dairy cow feces samples from Hebei Province, China). This strain possesses the DG9 gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. Simultaneously, this strain possesses the DG10 gene, the nucleotide sequence of which is shown in SEQ ID NO: 2, and refers to the following... Δdg9 The method for constructing the insect strain was completed simultaneously. Δdg10 Construction of insect strains.

[0047] 2. CRISPR knockout plasmid pCRISPR-U6- Δdg9 / Δdg10 Construction (1) gRNA primer design The DG9 gRNA sequences were designed using the EuPaGDT online tool (http: / / grna.ctegd.uga.edu / ): gRNA1 sequence is 5'-AAAGTGAGTAGCATTAGTGG-3'; gRNA2 sequence is 5'-CTATTGTTAATTTAGAATTC-3'. The DG10 gRNA sequences were also designed using the EuPaGDT online tool (http: / / grna.ctegd.uga.edu / ): gRNA1 sequence is 5'-CCTTTCACAACTCATTATCA-3'; gRNA2 sequence is 5'-TGTGATGGTTGTGACGGTTG-3'.

[0048] Simultaneously, primers for constructing double sgRNA plasmids were designed and synthesized. The specific primer information is shown in Table 1 below.

[0049] Table 1 Primers required for constructing the double sgRNA plasmid pCRISPR-U6-Δdg9 / Δdg10

[0050] (2) Construction of single gRNA CRISPR plasmid Using pACT1::Cas9-GFP-U6::sgTK plasmid as a template, based on the principle of homologous recombination, the designed gRNA sequence was added to the linearized primers of the plasmid, and the plasmid was linearized and amplified to construct a single sgRNA CRISPR plasmid. The PCR reaction system and procedure are shown in Tables 2 and 3 below.

[0051] Table 2 PCR reaction system

[0052] Table 3 PCR reaction procedure

[0053] Using restriction enzymes from Takara Corporation Dpn The PCR product was digested using I(1235S) to remove the plasmid template. The digestion reaction system is shown in Table 4 below.

[0054] Table 4 DpnI Reaction System

[0055] Mix the reaction solution thoroughly, centrifuge briefly, inactivate the PCR amplification enzyme at 70°C, then digest at 37°C for 20 min. Afterwards, use the EasyPure whole-gold PCR product purification kit. ® The digested PCR products were recovered using the PCRPurification Kit (EP101-01), and the concentration was then determined using a NanoDrop 2000 nucleic acid and protein analyzer from Thermo Fisher Scientific. The ligation was then performed using the ClonExpress II One-Step Cloning Kit (C112-01) from Novizan. The ligation system is shown in Table 5 below.

[0056] Table 5 Single-segment connection system

[0057] Where X represents the amount of vector plasmid used, specifically (0.02 × number of vector bases) ng / vector recovery concentration. Mix the reaction solution thoroughly, briefly centrifuge, and ligate at 37℃ for 30 min. After ligation, immediately place on ice and then transform into DH5α competent cells to construct the plasmid. In this process, the CRISPR plasmid containing gRNA1 is named pCRISPR-U6-DG9-6HA or pCRISPR-U6-DG10-smHA, and the CRISPR plasmid containing gRNA2 is named pCRISPR-U6- Δdg9 -g2 or pCRISPR-U6- Δdg10-gRNA2.

[0058] (3) Construction of dual gRNA CRISPR plasmids Using pCRISPR-U6-DG9-6HA plasmid as a template, PCR amplification of the plasmid backbone was performed using the primers grnaF-short / grnaR-short listed in Table 1. The PCR reaction conditions and procedures are shown in Tables 2 and 3. Then, using pCRISPR-U6- D dg9 Using plasmid -g2 as a template, the gRNA fragment was amplified using the primers gRNAf-long / gRNAr-long in Table 1. The PCR reaction conditions and procedure were the same as above. The PCR products were then detected by 0.8% agarose gel electrophoresis. The two correctly identified fragments were ligated using the Novizan ClonExpress II OneStep Cloning Kit (C112-01) to construct the plasmid, following the same construction method. The constructed plasmid was named pCRISPR-U6-Δdg9. Similarly, plasmid pCRISPR-U6-Δdg10 was constructed using the same method.

[0059] 3. Construction of homology repair plasmid pd-Δdg9 / Δdg10-tdTomato-Nluc-P2A-neo (1) Primer design for pd-Δdg9 / Δdg10-tdTomato-Nluc-P2A-neo plasmid Using the genomic DNA of the *Cryptospora microsporum* subtype IIdA20G1 from Hebei Province as a template, two homologous arms of the dg9 gene were specifically amplified: a 329 bp fragment from the 5'UTR region of the dg9 gene was selected upstream, and a 337 bp fragment from its 3'UTR region was used downstream; these two homologous arm fragments of dg9 were selected from those in Table 6. Δdg9 -5H-F / Δdg9-5H-FR and Δdg9 Amplification was performed using two pairs of specific primers: -3H-F / Δdg9-3H-F. Then, using the pIb-tdTomato-Nluc-P2A-neo plasmid as a template, the tdTomato-Nluc-P2A-neo fragment was amplified using the actin-F / 3utr-R primers in Table 6. The vector backbone containing the ampicillin resistance gene was amplified by PCR using the p5520-5510-2-6HA-Nluc-P2A-neo recombinant plasmid as a template. The primer sequences for amplifying the corresponding target fragments are shown in Table 6 below, and the specific amplification system and procedure are the same as those shown in Tables 2 and 3.

[0060] Similarly, using the genomic DNA of the *Cryptospora microsporum* subtype IIdA20G1 from Hebei as a template, two homologous arms of the dg10 gene were specifically amplified: a 400 bp fragment from the 5'UTR region of the dg10 gene was selected upstream, and a 480 bp fragment from its 3'UTR region was used downstream. These two homologous arm fragments of dg10 were selected from those shown in Table 6. Δdg10 -5H-F / Δdg10-5H-FR and Δdg10 Amplification was performed using two pairs of specific primers: -3H-F / Δdg10-3H-F, with the remaining methods being the same.

[0061] Table 6 Primers required for constructing homologous template plasmids

[0062] (2) PCR amplification and identification of plasmid fragments, recovery, ligation and transformation, colony screening and plasmid extraction The method and steps are the same as described above, and the homology repair plasmid pd- was successfully constructed and obtained. Δdg9 -tdTomato-Nluc-P2A-neo, plasmid pd- Δdg10 -tdTomato-Nluc-P2A-neo, plasmid stored at -20℃ for later use.

[0063] 4. Cryptosporidium microsporidium gene deletion strain Δdg9 Construction (1) Obtaining oocysts of Cryptosporidium microsporidium subtype IIdA20G1 strain from Hebei A magnetic stir bar and a fecal sample containing oocysts of Cryptosporidium megaterium (Hebei IIdA20G1 subtype) were placed together in a blue-mouthed bottle. The bottle was then placed on the magnetic stir bar and stirred at 4°C for 3 hours. Once no particulate feces remained, the sample was sieved, first through a 20-mesh sieve, then through a 60-mesh sieve. The sieved fecal liquid was then centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the sample was resuspended in 1 L of pure water. The mixture was then centrifuged again at 4000 rpm for 10 minutes, the supernatant was discarded, and the sample was resuspended in 1 L of pure water. The mixture was allowed to stand for 8 minutes to remove larger impurities.

[0064] After sedimentation, the supernatant was collected and centrifuged again. The supernatant was discarded, and the mixture was resuspended in 160 mL of pure water. Two pre-prepared solutions were then used: a 1:4 sucrose solution (65 mL saturated sucrose solution + 255 mL pure water) and a 1:2 sucrose solution (110 mL saturated sucrose solution + 220 mL pure water). Sixteen 50 mL centrifuge tubes were prepared. 20 mL of the 1:4 sucrose solution was added to each tube. Then, using a syringe, 20 mL of the 1:2 sucrose solution was slowly added from the bottom. Clear stratification was observed during this process. The suspension from the previous step was then slowly added to the top of the sucrose solution. The tubes were centrifuged at 1000 g for 25 min. The top 20 mL of waste liquid was discarded. Then, 20 mL of the oocyst suspension was transferred to a 250 mL centrifuge bottle. Pre-cooled pure water was added at a 1:1 ratio, and the tubes were centrifuged at 4000 rpm for 10 min. Finally, the suspension was resuspended in 5 mL of pre-cooled pure water to obtain the oocyst suspension.

[0065] Finally, a cesium chloride density gradient centrifugation was performed. 1 mL of cesium chloride was added to a 1.5 mL low-adsorption centrifuge tube, and then 500 μL of the oocyst suspension was gently added to the upper layer of the cesium chloride solution using a pipette. The mixture was centrifuged at 13200 rpm for 3 min. After centrifugation, a white oocyst band was visible at the 1 mL mark. 700 μL of this band was added to a 15 mL low-adsorption centrifuge tube, followed by the addition of pre-chilled pure water at a 1:1 ratio. The mixture was centrifuged at 10000 g for 10 min, the supernatant was discarded, and the washing was repeated twice. Finally, the oocysts were resuspended in 1 mL of pre-chilled PBS, mixed well, and triple antibody was added at a 1:100 ratio. The cells were counted using a hemocytometer and stored at 4°C.

[0066] (2) Obtaining fresh sporozoites of Cryptosporidium microsporidium subtype IIdA20G1 strain from Hebei Absorb 2.5×10 7 Fresh oocysts were placed on an ice pack, and 200 μL of Clorox sterilizing solution and 600 μL of PBS were added. The mixture was incubated on ice for 10 min. The oocysts were centrifuged at 13200 rpm for 3 min at 4°C, and the supernatant was discarded completely. The oocysts were washed three times with PBS. The oocysts were then resuspended in 400 μL of 1% BSA, followed by the addition of 400 μL of 1.5% cholesulfonate to a final cholesulfonate concentration of 0.75%. The mixture was incubated in a 37°C water bath for 60 min. After decysting, the oocysts were centrifuged at 13200 rpm for 3 min and the supernatant was discarded. The sporozoite suspension was then resuspended in 1 mL of room temperature PBS, centrifuged at 13200 rpm for 3 min, and the supernatant was discarded. This process was repeated twice.

[0067] (3) Sporopolation and GKO mouse gavage infection Using the 4D nuclear transfection kit (SFCellLine4D-NucleofectorTMXKitL, V4XC-2024) from Lonza, Germany, sporozoites were resuspended in 80 μL of electroporation buffer (composed of 65.6 μL LSF buffer and 14.4 μL L1 buffer). The specific reaction solution preparation is shown in Table 7 below. The required amount of both the CRISPR knockout plasmid and the homology repair plasmid is 50 μg, and their concentration needs to be adjusted to 5000 ng / μL before electroporation.

[0068] Table 7. 100µL Electrophoresis System

[0069] The mixed liquid was transferred to an electroporation vessel and electroporated using the AMAXA4D-Nucleofector system from Lonza, Germany, via the EH100 program. The electroporated sporozoites were diluted with 200 µL of PBS and incubated at room temperature.

[0070] (4) GKO mice were infected by gavage Three 3-5 week old GKO mice (IFN-γ knockout mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and bred at the Experimental Animal Center of South China Agricultural University) were prepared in advance. Each mouse was gavaged with 200 μL of 8% NaHCO3 solution to neutralize gastric acid. Five minutes later, each mouse was gavaged with 100 μL of PBS solution containing electroporated sporozoites. Twenty-four hours after gavage, the GKO mice were screened for drug efficacy in vivo using 16 g / L paromomycin sulfate solution instead of ordinary drinking water.

[0071] (5) Detection of luciferase value Subsequent monitoring of Cryptosporidium oocyst release was conducted by detecting luciferase activity in the feces of infected mice. Fresh fecal samples were collected from mice at 4, 9, and 14 days after infection and placed in 1.5 mL centrifuge tubes. 8–10 3 mm glass beads and 0.8 mL of fecal lysis buffer were added, and the mixture was shaken for 1 min, followed by centrifugation at 19000 g for 1 min. 50 μL of the supernatant was added to an ELISA plate, and a 25:1 mixture of luciferase substrate buffer and luciferase substrate (Promega Nano-Glo Luciferase Kit, N1120) was prepared. The sample was then incubated at room temperature in the dark for 3 min. Finally, the Nluc value was measured using a multi-functional microplate reader (Berten Instruments, Inc.). Once the mouse feces tested positive for luciferase, fecal samples were collected daily to obtain more Cryptosporidium oocysts and to obtain a purified gene-edited strain. Δdg9 Insect strains.

[0072] Similarly, obtain the results using the methods described above. Δdg10Insect strains.

[0073] (6) Δdg9、Δdg10 Identification of insect strains PCR1, PCR2 and PCR3 primers were designed and synthesized to identify the purified gene-edited insect strains. The specific primer sequences are shown in Table 8 below. The reaction system and procedure for PCR detection are the same as those shown in Tables 2 and 3.

[0074] Table 8. Primers for PCR identification

[0075] A schematic diagram illustrating the construction principles of different gene-deleted insect strains is shown below. Figure 1 As shown in the results, the presence of bands in PCR1 and PCR2 indicates that the tdTomato fluorescent tag, the drug selection fragment Neo, and the luciferase fragment Nluc have been integrated into the Cryptosporidium microsporidium DG9 site. The PCR identification results are as follows: Figure 2 As shown, it indicates Δdg9 The insect strain was successfully constructed. Similarly, for... Δdg10 The insect strain was tested and identified, and the results showed... Δdg10 The insect strain was successfully constructed.

[0076] Example 2 Cryptosporidium microphyllum Δdg9Δdg10 Construction of double knockout strains 1. CRISPR knockout plasmid pCRISPR-U6- Δdg9Δdg10 Construction (1) gRNA primer design The gRNA sequences selected in this experiment were 5'-CTATTGTTAATTTAGAATTC-3' and 5'-TGTGATGGTTGTGACGGTTG-3', the former being the same as the Δdg9-gRNA2 gRNA sequence and the latter being the same as the pCRISPR-U6-DG10-smHAgRNA1 sequence.

[0077] (2) pCRISPR-U6- Δdg9Δdg10 Plasmid construction and transformation, colony screening and plasmid extraction Using the pCRISPR-U6-Δdg9-gRNA2 and pCRISPR-U6-DG10-smHA-gRNA1 plasmids constructed in Example 1 as templates, the gRNA sequence was amplified. The primer sequences were the same as in Table 1, and the methods and steps were the same as in Example 1. The CRISPR knockout plasmid pCRISPR-U6- was successfully constructed. Δdg9Δdg10 The plasmid should be stored at -20℃ for later use.

[0078] 2. Homologous repair plasmid pd- Δdg9Δdg10 Construction of -tdTomato-Nluc-P2A-neo Using the whole genome of *Cryptospora microsporum* subtype Hebei IIdA20G1 as a template, the 5'UTR of DG10 was selected as the left homologous arm, and the 3'UTR of DG9 as the right homologous arm, with fragment lengths of 400 bp and 337 bp, respectively. Primers Δdg10-5H-F / Δdg10-5H-R and Δdg9-3H-F / Δdg9-3H-R, as listed in Table 6, were used. The left and right homologous arm fragments, the tdTomato-Nluc-P2A-neo fragment, and the Amp backbone were all obtained using the method in Example 1. The above four fragments were ligated, transformed, screened for colonies, and extracted into plasmids, following the same steps as in Example 1, to obtain the homology repair plasmid pd- Δdg9Δdg10 The constructed plasmid, -tdTomato-Nluc-P2A-neo, was stored at -20°C for subsequent experiments.

[0079] 3. Cryptosporidium microsporidium gene deletion strain Δdg9Δdg10 Construction The dual sgRNA CRISPR plasmid pCRISPR-U6- Δdg9Δdg10 With homologous recombination plasmid pd- Δdg9Δ dg10 -tdTomato-Nluc-P2A-neo was co-transfected into wild-type Cryptosporidium microsporidium strains using the same method as in Example 1. After drug screening and PCR identification, the results are as follows: Figure 2 As shown, successful identification was obtained. Δdg9Δdg10 The insect strain obtained was Cryptosporidium microsporidium DG9DG10 double gene deletion strain.

[0080] Example 3 Cryptosporidium microphyllum Δdg9、Δdg10 In vitro growth experiment of insect strains In a sample infected with a gene-edited parasite strain, the luciferase level showed a linear relationship with the number of parasites, increasing with the increase in parasite number. Since Cryptosporidium's proliferation time after invading host cells is fixed, the effect of gene editing on parasite proliferation can be roughly inferred by comparing luciferase expression levels at different time points. If gene deletion affects parasite proliferation, a slowdown in parasite proliferation may be observed at specific time points (developmental stages associated with the deleted gene). This example assesses the growth status of the gene-edited parasite strain through in vitro experiments.

[0081] 1. In vitro infection The samples obtained using Example 1 were used respectively. Δdg9、Δdg10HCT-8 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, CBP60030) were infected with oocysts of the parasite strain and cultured in 2% FBS1640 complete medium. Once the cell confluence reached more than 60%, Cryptosporidium was inoculated. On 48-well cell culture plates, fresh oocysts were first aspirated and placed on an ice box, then 200 μL of Clorox sterile water and 600 μL of PBS were added, and the plates were incubated on ice for 10 min. After that, the plates were centrifuged at 13200 rpm for 3 min at 4°C, and the supernatant was completely discarded. The oocysts were washed three times with PBS. Finally, the oocysts were resuspended in 2% FBS 1640 medium in a clean bench, while the old HCT-8 cell culture medium was discarded. The resuspended oocyst suspension was then added to HCT-8 cells. When the HCT-8 cell confluence reached more than 80%, 20,000 oocysts were infected per well. At the same time, the wild-type Cryptosporidium microsporidium strain DG9-6HA (derived from the DG9 genetic marker strain constructed at the same time in this experiment) was used as a control (with drug screening tag and Nluc fluorescent tag).

[0082] 2. Luciferase assay Each experimental group had four replicates, and the luciferase values ​​of each well were measured at 3, 12, 24, 36, and 48 hours post-infection. The luciferase assay method was the same as in Implementation 1. After the assay was completed, the data were statistically analyzed using GraphPad 9.0.

[0083] Experimental results are as follows Figure 3 As shown, the DG9 gene deletion group showed no significant difference from the control group in the early stages of infection (3h, 12h, 24h), but exhibited significant growth inhibition in the later stages of infection (36h, 48h). P <0.05. The DG10 gene knockout group showed no significant difference from the control group in the early stages of infection (3h and 12h), but exhibited significant growth differences in the later stages of infection (24h, 36h, and 48h). P <0.05). The results indicate that the loss of DG9 and DG10 proteins significantly affects the growth and development process of Cryptosporidium microsporidium in the later stages of in vitro infection.

[0084] Example 4 Cryptosporidium microphyllum Δdg10 In vivo infection experiment of insect strain In vivo infection experiments are an important method for assessing the pathogenicity of Cryptosporidium microsporidium. This study systematically monitored key indicators such as the amount of oocysts excreted after infection, the development of disease symptoms, and changes in survival rate.

[0085] 1. Test treatment The experiment used 3-5 week old GKO mice, with each mouse housed individually and the mean weight of each group being kept approximately the same. A Δdg10 group was established: each mouse was inoculated with 1×10⁻⁶ g of vaccine. 4 indivual Δdg10Fresh egg sacs of the insect strain were collected; a control group consisting of wild-type Cryptosporidium microsporidium was set up: the DG10-smHA strain (derived from the DG10 genetic marker strain constructed concurrently in this experiment) was also inoculated with 1×10⁻⁶ eggs. 4 Fresh oocysts were administered to mice; a control group was also included, in which no oocysts were inoculated. All mice were treated with paromomycin (16 g / L) during the experiment.

[0086] 2. Comparison of oocyst excretion intensity Oocyst excretion intensity was determined by the luciferase value of fresh feces. Fresh feces were collected every two days starting from the second day after infection to determine the luciferase value of fresh feces. The luciferase determination method was the same as in Example 1, and the change curve of oocyst excretion intensity was plotted using GraphPad 9.0 software.

[0087] The results are as follows Figure 4 As shown, the amount of oocysts excreted in mice after infection with the parasite strain continued to increase, peaking on day 12. (Comparison) D dg10 The dynamic curves of oocyst excretion in the DG10-smHA group and the DG10-smHA group showed no significant difference, indicating that the deletion of the dg10 gene did not significantly change the proliferation efficiency of Cryptosporidium microsporidium in the host.

[0088] 3. Comparison of changes in mouse body weight To assess the impact of DG10 protein deficiency on the pathogenicity of Cryptosporidium parvum, this study dynamically monitored changes in body weight in infected mice. Body weight was measured in both the experimental and control groups before infection and every two days after infection. Results are as follows: Figure 5 As shown, both groups showed a weight loss trend starting from day 6 of infection, with the DG10-smHA group exhibiting a slightly higher rate of weight loss. Δdg10 The two groups showed similar overall decline rates in the later stages of infection.

[0089] 4. Comparison of mouse survival rates The results of dynamic monitoring of the survival status of infected mice are as follows: Figure 6 As shown, the first death in the DG10-smHA group occurred on the 12th day after infection, followed by 1 and 2 more deaths on the 20th and 26th days after infection, respectively, with the final cumulative mortality rate reaching 100%. Δdg10 The first deaths in the group occurred on day 20 post-infection (2 cases), and the overall survival rate was 50% by the experimental endpoint (day 33 post-infection). Data showed that mice infected with the DG10 protein-deficient strain had a longer survival period and a higher survival rate, suggesting that DG10 deficiency may weaken the pathogenicity of the parasite.

[0090] 5. Comparison of clinical symptoms in mice By recording and comparing the clinical symptoms of the two groups of mice, dynamic monitoring results showed that the DG10-smHA-labeled group of mice began to show typical clinical symptoms on day 8 post-infection, while Δdg10 The onset of clinical symptoms in the deletion group was delayed until day 14 post-infection, 6 days later than in the labeled group. At the peak of infection, both groups exhibited typical symptoms such as lethargy, ruffled fur, arched back, reduced movement, and severe diarrhea, but the clinical manifestations were more pronounced in the labeled group. Figure 7 As shown in the figure, assessment based on the clinical symptom scoring criteria revealed that the marked group scored significantly higher than the deletion group. Combined with the results of weight change and survival analysis, this suggests that DG10 deletion reduces the infectivity of Cryptosporidium microsporidium, exhibiting a significant attenuation effect.

[0091] Example 5: In vivo infection experiment of Cryptosporidium microsporidium strain with double deletion of DG9 and DG10 proteins. To investigate the effects of single deletion of the DG9 protein and double deletion of both DG9 and DG10 proteins on the pathogenicity of Cryptosporidium, this study conducted animal infection experiments. Twenty IFN-γKO mice were used and divided into four groups of five each. The mice were infected via gavage with a dg9 gene-marked strain (DG9-6HA group), a dg10 gene-deleted strain, and a double deletion of both dg9 and dg10 genes, respectively. Clinical symptoms and mortality rates were closely monitored to assess the roles of the dg9 and dg10 genes in Cryptosporidium pathogenicity.

[0092] 1. Comparison of oocyst excretion intensity We inoculated five IFN-γKO mice with each of the three parasite strains via gavage, with an infection dose of 1 × 10⁻⁶ per mouse. 4 One oocyst was produced. The oocyst excretion intensity was assessed by detecting NLUC values, and the proliferation of different strains in mice was then analyzed. Experimental results are as follows: Figure 8 As shown, the oocyst excretion intensity of mice in all three groups showed a continuous upward trend after infection, until reaching a peak. Comparative analysis showed that the DG9-6HA group, Δdg9 Groups and Δdg9Δdg10 There was no significant difference in excretion intensity among the groups, indicating that the deletion of the dg9 gene alone or the simultaneous deletion of the dg9 and dg10 genes did not significantly affect the in vivo proliferation capacity of Cryptosporidium microlucum.

[0093] 2. Comparison of changes in mouse body weight To investigate the effects of DG9 protein deletion and dual deletion of DG9 and DG10 proteins on the pathogenicity of Cryptosporidium parvum, we measured the body weight of mice before and every two days after infection and recorded the changes in body weight. The experimental results are as follows: Figure 9 As shown, on day 6 post-infection, the body weight of mice in all three groups began to decrease. Among them, the mice infected with the DG9-6HA strain experienced the fastest weight loss, while... Δdg9 Groups and Δdg9Δdg10 The mice in the double-deletion group experienced a similar rate of weight loss, which was slow.

[0094] 3. Comparison of mouse survival rates Mouse survival curves as follows Figure 10 As shown, there were significant differences in the survival of mice infected with different strains of the parasite. The first mouse in the DG9-6HA group died on day 12 post-infection, followed by one death each on days 13, 15, 18, and 24 post-infection. D dg9 The mice in the group began to die on day 18 after infection, with one mouse dying first, followed by one mouse dying on day 24, two mice dying on day 26, and one mouse dying on day 34. Δdg9Δdg10 The first mouse death occurred on day 22 post-infection, followed by two deaths on day 24, and one death each on days 25 and 30. Statistical analysis showed that the survival rate of mice in the DG9-6HA group was significantly higher than that in the DG9-6HA group. D dg9 Groups and Δdg9Δdg10 Significant differences were found in all groups ( P <0.05), combined with the above experimental results, it shows that the deletion of dg9 or dg10 genes alone, as well as the double deletion of dg9 and dg10 genes, can significantly prolong the survival time of mice.

[0095] 4. Comparison of clinical symptoms in mice Through continuous observation and comparative analysis of the clinical symptoms of the three groups of mice, significant differences in symptom presentation were found among mice infected with different strains of the parasite. Mice in the DG9-6HA group were the first to develop clinical symptoms on day 8 post-infection, while... Δdg9 Groups and D dg9Δdg10 Mice in the DG9-6HA group did not show symptoms until 10 days after infection. In terms of symptom development, the DG9-6HA group mice experienced faster symptom progression, while the Δdg9 and Δdg9Δdg10 groups showed relatively slower symptom changes. At the peak of infection, such as... Figure 11 As shown, mice in the DG9-6HA group exhibited severe arched backs, rough and disheveled fur, and emaciation; in contrast, mice in the Δdg9 and Δdg9Δdg10 groups had smooth fur and were more active. According to the clinical symptom scoring criteria, the scores of the three groups of mice are as follows: Figure 11 As shown, the scores of mice in the DG9-6HA group were significantly higher than those in the DG9-6HA group. Δdg9 Groups and Δdg9Δdg10 The scores of the two groups were not significantly different.

[0096] Based on previous experimental results, it is shown that the deletion of the dg9 or dg10 gene alone, as well as the double deletion of both dg9 and dg10 genes, can effectively reduce the virulence of Cryptosporidium microsporum. Furthermore, the genetic background of strains with double deletion of both dg9 and dg10 genes is more stable, with a significantly reduced risk of reversion mutations, resulting in higher reliability and controllability in subsequent applications of attenuated vaccines.

[0097] In summary, this invention, utilizing CRISPR / Cas9 gene editing technology, successfully constructed DG9 or DG10 and their double-gene deletion strains for the first time. Using a marker strain as a control, the effects of DG9 and DG10 on host pathogenicity were systematically evaluated through in vitro HCT-8 cell infection experiments and in vivo animal experiments. In vitro experimental results showed that, compared with the control group, the deletion of DG9 and DG10 significantly inhibited the in vitro proliferation of Cryptosporidium microsporum. Combined with life history analysis, it is speculated that DG9 and DG10 begin to regulate the growth and development of the parasite during the schizont stage, possibly inhibiting parasite reproduction by affecting intracellular developmental processes. Infection of IFN-γ-deficient mice with DG9, DG10, or double-deletion strains resulted in significantly reduced clinical symptoms, delayed mortality, and prolonged survival. Furthermore, the gene-deleted strains exhibited weakened virulence, lower parasite load in mice, and reduced oocyst excretion, demonstrating reduced virulence. Simultaneously, the double-deletion strains showed a more stable genetic background and a significantly reduced risk of reversion mutations, resulting in higher reliability and controllability in subsequent attenuated vaccine applications. This facilitates the development of new drug targets or the preparation of gene knockout strains as candidate attenuated vaccines, laying a solid foundation for the control of Cryptosporidium microsporidium.

[0098] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Cryptosporidium microsporidium gene-deleted strain, characterized in that, The strain was obtained by directly knocking out the DG9 and / or DG10 genes of Cryptosporidium microsporum dense granule protein using gene editing technology; the nucleotide sequence of the DG9 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the DG10 gene is shown in SEQ ID NO:

2.

2. The method for constructing the Cryptosporidium microsporidium gene-deleted strain according to claim 1, characterized in that, Includes the following steps: S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as a template, construct double sgRNA CRISPR plasmids pCRISPR-U6-Δdg9 or pCRISPR-U6-Δdg10 using DG9 or DG10 gene sgRNA primers. S2. Using pIb-tdTomato-Nluc-P2A-neo plasmid and wild-type Cryptosporidium microsporum DNA as templates, construct homologous recombinant plasmids pd-Δdg9-tdTomato-Nluc-P2A-neo or pd-Δdg10-tdTomato-Nluc-P2A-neo plasmids using DG9 or DG10 gene primers; S3. The double sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into wild-type Cryptosporidium microsporidium strains. Drug screening and PCR identification were then performed to obtain Cryptosporidium microsporidium strains with DG9 or DG10 gene deletions. Δdg9 or Δdg10 ; or S11. Using pCRISPR-U6- Δdg9 The pCRISPR-U6-gRNA2 plasmid, pCRISPR-U6-gRNA1 plasmid, and the homologous recombinant plasmid pCRISPR-U6-gRNA1 plasmid were constructed using the whole genome of Cryptosporidium microsporum as a template and DG9DG10 gene primers. Δdg9Δ dg10 ; S21. Using pIb-tdTomato-Nluc-P2A-neo plasmid and wild-type Cryptosporidium microsporum DNA as templates, and employing DG9DG10 gene primers, construct the homologous recombinant plasmid pd- Δdg9Δdg10 -tdTomato-Nluc-P2A-neo; S31. The double sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into wild-type Cryptosporidium microsporidium strains. The DG9DG10 double gene deletion strain of Cryptosporidium microsporidium was obtained through drug screening and PCR identification. Δdg9Δdg10 .

3. The construction method according to claim 2, characterized in that, In step S1, using pCRISPR-U6-DG9-6HA and pCRISPR-U6-Δdg9-g2 plasmids as templates, and employing DG9 gene sgRNA primers, a double sgRNA CRISPR plasmid pCRISPR-U6-Δdg9 is constructed; using pCRISPR-U6-DG10-smHA and pCRISPR-U6-Δdg10-gRNA2 plasmids as templates, and employing DG10 gene sgRNA primers, a double sgRNA CRISPR plasmid pCRISPR-U6-Δdg10 is constructed.

4. The construction method according to claim 2, characterized in that, The primer sequences for the DG9 or DG10 gene sgRNA in step S1 are shown in SEQ ID NO: 3~8.

5. The construction method according to claim 2, characterized in that, The primer sequences for the DG9 or DG10 gene in step S2 are shown in SEQ ID NO: 9~20.

6. The construction method according to claim 2, characterized in that, The wild-type Cryptosporidium microsporidium strain is the Hebei IIdA20G1 subtype of wild-type Cryptosporidium microsporidium.

7. The application of reagents for deleting or knocking out the Cryptosporidium DG9 and / or DG10 genes in the construction of DG9 and / or DG10 gene-deleted strains or in the preparation of Cryptosporidium vaccines, characterized in that, The nucleotide sequence of the DG9 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the DG10 gene is shown in SEQ ID NO:

2.

8. The application of Cryptosporidium DG9 and / or DG10 genes as targets in the preparation of Cryptosporidium gene-deleted strains or in the preparation of Cryptosporidium vaccines, characterized in that... The DG9 and / or DG10 genes are deleted or knocked out in Cryptosporidium microlucum; the nucleotide sequence of the DG9 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the DG10 gene is shown in SEQ ID NO:

2.

9. The use of the Cryptosporidium microsporidium gene-deleted strain according to claim 1 in the preparation of Cryptosporidium microsporidium vaccine.

10. A Cryptosporidium parvum vaccine, characterized in that, The strain containing the Cryptosporidium microsporidium gene deletion as described in claim 1.

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