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CN-122012489-A - Screening method of targeting amino acid chelated zinc metabolic genes based on Crispr whole genome library

CN122012489ACN 122012489 ACN122012489 ACN 122012489ACN-122012489-A

Abstract

The invention discloses a screening method of targeting amino acid chelated zinc metabolic genes based on a Crispr whole genome library. The method comprises the steps of constructing a single-gene knockout cell strain of the intestinal epithelial cells with zinc transporter gene knockout, transfecting a Crispr whole genome knockout library into the single-gene knockout cell strain of the intestinal epithelial cells to obtain double-gene knockout cell strains of the intestinal epithelial cells, treating the double-gene knockout cell strains of the intestinal epithelial cells by using different zinc sources, dyeing by using fluorescent dyes, sorting the double-gene knockout cell strains of the intestinal epithelial cells with zinc transport difference by using a flow cytometry, extracting gDNA (global DNA) from the sorted cell lines, amplifying gRNA sequences, sequencing the gRNA sequences in a high throughput manner, and identifying key target genes for regulating and controlling the metabolism of the zinc chelate of different amino acids by using bioinformatics analysis. The invention establishes a method for screening amino acid chelated zinc specific metabolic genes in high throughput, and simultaneously screens and obtains Zn metabolic regulation gene libraries.

Inventors

  • WAN DAN
  • Gong Chengyan
  • DONG YACHAO
  • YIN YULONG

Assignees

  • 中国科学院亚热带农业生态研究所

Dates

Publication Date
20260512
Application Date
20251217

Claims (10)

  1. 1. A screening method of targeting amino acid chelated zinc metabolic genes based on Crispr whole genome library. The screening method is characterized by comprising the following steps of: (1) Constructing a single gene knockout cell strain of intestinal epithelial cells which is defective in zinc transport function and can stably express Cas9 by using a Crispr-Cas9 technology, wherein the single gene is ZIP4, ZIP5 or ZIP14 related to the zinc transport function; (2) Carrying out slow virus packaging on Crispr whole genome knockout library plasmids, and then transfecting the plasmids into the intestinal epithelial cell gene knockout cell strain constructed in the step (1) to obtain an intestinal epithelial cell double-gene knockout cell library for stably expressing Cas 9; (3) Treating the intestinal epithelial cell double-gene knockout cell library constructed in the step (2) with at least one zinc source, then staining intestinal epithelial cell double-gene knockout cell strains in the intestinal epithelial cell double-gene knockout cell library with a fluorescent dye sensitive to zinc ions, and separating out a zinc transport difference intestinal epithelial cell double-gene knockout cell library with difference in intracellular zinc concentration by a flow cytometry; (4) Extracting whole genome DNA of the zinc transport difference intestinal epithelial cell double-gene knockout cell library selected in the step (3), and amplifying the gRNA sequence of the zinc transport difference intestinal epithelial cell double-gene knockout cell line by taking the whole genome DNA as a template; (5) Comparing the gRNA sequence obtained in the step (4) with the gRNA sequence of the Crispr whole genome through bioinformatics analysis, and identifying genes enriched in cells with weak zinc transport capacity in a zinc transport difference intestinal epithelial cell double-gene knockout cell line, wherein the genes are targeted amino acid chelated zinc metabolic genes.
  2. 2. The method of screening for a zinc amino acid chelate metabolic gene based on a Crispr whole genome library according to claim 1, wherein the monogen in step (1) is ZIP4 or ZIP5.
  3. 3. The method for screening a whole genome library targeted amino acid chelated zinc metabolic gene based on Crispr according to claim 1, wherein the intestinal epithelial cells in step (1) are colon cancer epithelial cells CaCO2 or pig jejunum epithelial cells IPEC-J2.
  4. 4. The screening method of zinc metabolism genes based on the targeting amino acid chelate of a whole genome library of Crispr as claimed in claim 1, wherein the step (1) is to design gRNA for genes related to zinc transport function, construct knock-out plasmids containing Crispr-Cas9 system and gRNA by using the designed gRNA, simultaneously transfect the constructed knock-out plasmids and commercial lentiviral packaging plasmids into 293T cells, collect lentiviral supernatant after 3-4d, add the collected lentivirus into target cells containing genes related to zinc transport function, continue to culture the target cells, and obtain target gene knock-out cell strains for stably expressing Cas9 cells after blasticidin screening.
  5. 5. The method for screening the targeting amino acid chelated zinc metabolic gene based on the Crispr whole genome library according to claim 1, wherein the step (2) is to perform slow virus packaging on the plasmid of the human whole genome knockout library, then transfect a single gene knockout cell strain, culture, and screen by puromycin to obtain a double gene knockout cell library for stably expressing Cas 9.
  6. 6. The screening method of the amino acid chelated zinc metabolic gene based on the Crispr whole genome library according to claim 1, wherein in the step (3), the zinc source is one or more of amino acid chelated zinc, the zinc concentration is 100 mu M, the zinc source treatment time is 6 hours, the fluorescent dye is Zinpy-1 dye, the dyeing time is 30min, and the flow cytometer sorting is performed at least three times.
  7. 7. The method for screening a targeting amino acid chelated zinc metabolic gene based on a Crispr whole genome library according to claim 1, wherein the targeting amino acid chelated zinc metabolic gene in step (5) is CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, ZNF570.
  8. 8. The use of the targeted amino acid chelated zinc metabolic gene of claim 7 in targeted design of new trace element feed additives.
  9. 9. Use of the targeted amino acid chelated zinc metabolic gene of claim 7 in the preparation of a feed additive for improving animal nutritional status or in the preparation of a nutritional supplement for improving human zinc nutritional status or in the preparation of a medicament for treating human zinc metabolism related diseases.
  10. 10. The Zn metabolism regulating gene library is characterized in that genes in the Zn metabolism regulating gene library are CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRAP 10-5, GNAT1 and ZNF570.

Description

Screening method of targeting amino acid chelated zinc metabolic genes based on Crispr whole genome library Technical Field The invention belongs to the technical field of genetic engineering and functional genomics, and particularly relates to a screening method of targeting amino acid chelated zinc metabolic genes based on a Crispr whole genome library. Background Zinc is used as trace element necessary for animals and human beings, and plays a core role in various physiological processes such as organism growth, immune response, antioxidation stress, reproduction and the like. The functions are realized mainly by the structural composition and catalytic reaction of a plurality of enzymes, and the functions are used as key regulatory factors of transcription factors and signal paths. The metabolic homeostasis of zinc ions (Zn2+) in cells is mainly cooperatively regulated by three major protein families of Zip, znT and Metallothionein (MT), wherein the Zip family mediates the transport of zinc ions into cytoplasm, the ZnT family is responsible for discharging the zinc ions out of cytoplasm, and MT participates in fine regulation by reversibly combining the zinc ions. A large number of researches and production practices show that compared with inorganic zinc sources, organic zinc (such as amino acid chelated zinc, protein zinc, peptide zinc and the like) has higher absorptivity and bioavailability, and has obvious advantages in the aspects of promoting animal growth, enhancing immune function and reducing fecal zinc emission. At the same zinc addition level, the amino acid chelated zinc and the free Zn2+ have obvious difference in intestinal tract absorption and epithelial transport efficiency. The Crispr-Cas9 whole genome knockout library technology can perform functional deletion screening in the whole genome range, is a powerful tool for finding drug targets or key genes, and can perform high-throughput multi-parameter sorting on cells by flow cytometry. However, there is no report on the combination of the two technologies, which is specially used for screening amino acid chelated zinc metabolic genes. That is, the existing technology means with high efficiency and strong specificity is still lacking, so that the metabolic target genes of the amino acid chelated zinc are systematically analyzed, the deep research and development and the product popularization and application of the related technology are restricted to a certain extent, and the difficulty of market education is increased. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides a screening method of targeted amino acid chelated zinc metabolic genes based on a Crispr whole genome library. In order to achieve the above purpose, the technical scheme provided by the invention is as follows: the screening method of the targeting amino acid chelated zinc metabolic gene based on the Crispr whole genome library comprises the following steps: (1) Constructing a single gene knockout cell strain of intestinal epithelial cells which is defective in zinc transport function and can stably express Cas9 by using a Crispr-Cas9 technology, wherein the single gene is ZIP4, ZIP5 or ZIP14 related to the zinc transport function; (2) Carrying out slow virus packaging on Crispr whole genome knockout library plasmids, and then transfecting the plasmids into the intestinal epithelial cell gene knockout cell strain constructed in the step (1) to obtain an intestinal epithelial cell double-gene knockout cell library for stably expressing Cas 9; (3) Treating the intestinal epithelial cell double-gene knockout cell library constructed in the step (2) with at least one zinc source, then staining intestinal epithelial cell double-gene knockout cell strains in the intestinal epithelial cell double-gene knockout cell library with a fluorescent dye sensitive to zinc ions, and separating out a zinc transport difference intestinal epithelial cell double-gene knockout cell library with difference in intracellular zinc concentration by a flow cytometry; (4) Extracting whole genome DNA of the zinc transport difference intestinal epithelial cell double-gene knockout cell library selected in the step (3), and amplifying the gRNA sequence of the zinc transport difference intestinal epithelial cell double-gene knockout cell library by taking the whole genome DNA as a template; (5) Comparing the gRNA sequence obtained in the step (4) with the gRNA sequence of the Crispr whole genome through bioinformatic analysis, and identifying genes enriched in cells with weak zinc transport capacity in a zinc transport difference intestinal epithelial cell double-gene knockout cell library, wherein the genes are targeted amino acid chelated zinc metabolic genes. Preferably, the monogenic in step (1) is ZIP4 or ZIP5. Preferably, the intestinal epithelial cells in step (1) are colon cancer epithelial cells CaCO2 or pig jejunal epithelial cells IPEC-J2. Pre