Screening method for targeting zinc amino acid chelate metabolism gene based on Crispr whole genome library

Abstract

The invention discloses a screening method for targeted zinc amino acid chelate metabolism genes based on a Crispr whole genome library. The method comprises the following steps: constructing a zinc transporter gene knockout intestinal epithelial cell single gene knockout cell strain; transfecting the Crispr whole genome knockout library into an intestinal epithelial cell single gene knockout cell strain to obtain an intestinal epithelial cell double gene knockout cell strain; treating the intestinal epithelial cell double-gene knockout cell strain with different zinc sources, dyeing with a fluorescent dye, and sorting zinc transport difference intestinal epithelial cell double-gene knockout cell strains through flow cytometry; the separated cell lines are subjected to gDNA extraction, gRNA sequence amplification and high-throughput sequencing, and key target genes for regulating and controlling metabolism of different amino acid chelated zinc are analyzed and identified through bioinformatics. The invention establishes a method for high-throughput screening of the specific metabolism gene of the amino acid chelated zinc, and a Zn metabolism regulation gene library is also obtained through screening at the same time.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and functional genomics, specifically relating to a screening method for targeted amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library. Background Technology

[0002] Zinc, an essential trace element for animals and humans, plays a central role in various physiological processes, including growth, immune response, anti-oxidative stress, and reproduction. Its functions are primarily achieved through participation in the structural composition and catalytic reactions of various enzymes, as well as acting as a transcription factor and a key regulator of signaling pathways. Intracellular zinc ion (Zn²⁺) metabolic homeostasis is mainly regulated by the synergistic action of three major protein families: the Zip family mediates the transport of zinc ions into the cytoplasm, the ZnT family is responsible for their expulsion from the cytoplasm, and MT participates in fine regulation through reversible binding of zinc ions.

[0003] Numerous studies and production practices have shown that organic zinc (such as amino acid chelated zinc, protein zinc, and peptide zinc) has higher absorption rates and bioavailability compared to inorganic zinc sources, exhibiting significant advantages in promoting animal growth, enhancing immune function, and reducing fecal zinc emissions. At the same zinc supplementation level, amino acid chelated zinc and free Zn²⁺ show significant differences in intestinal absorption and epithelial transport efficiency. Crispr-Cas9 genome-wide knockout library technology can perform loss-of-function screening across the entire genome, making it a powerful tool for discovering drug targets or key genes; flow cytometry can perform high-throughput, multi-parameter sorting of cells. However, there are currently no reports of combining these two technologies specifically for screening amino acid chelated zinc metabolic genes. In other words, there is still a lack of efficient and highly specific technical means to systematically analyze the metabolic target genes of amino acid chelated zinc, which to some extent restricts the in-depth research and development of related technologies and the promotion and application of products, and increases the difficulty of market education. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a screening method based on CRISPR whole-genome library targeting amino acid chelation zinc metabolism genes.

[0005] To achieve the above objectives, the technical solution provided by this invention is as follows: The screening method based on the CRISPR whole-genome library targeting amino acid chelation zinc metabolism genes includes the following steps: (1) Construct a single-gene knockout cell line of intestinal epithelial cells that stably expresses Cas9 and has zinc transport function defects by using CRISPR-Cas9 technology; the single gene is ZIP4, ZIP5 or ZIP14, which are related to zinc transport function. (2) The Crispr whole genome knockout library plasmid was packaged with lentivirus and then transfected into the intestinal epithelial cell gene knockout cell line constructed in step (1) to obtain an intestinal epithelial cell double gene knockout cell library that stably expresses Cas9. (3) Treat the intestinal epithelial cell double gene knockout cell library constructed in step (2) with at least one zinc source, and then stain the intestinal epithelial cell double gene knockout cell line in the intestinal epithelial cell double gene knockout cell library with zinc ion sensitive fluorescent dye. Then sort the intestinal epithelial cell double gene knockout cell library with different zinc transport intracellular zinc concentration by flow cytometry. (4) Extract whole genome DNA from the zinc transport differential intestinal epithelial cell double gene knockout cell library sorted in step (3), and amplify the gRNA sequence of the zinc transport differential intestinal epithelial cell double gene knockout cell library using the whole genome DNA as a template. (5) By bioinformatics analysis, the gRNA sequence obtained in step (4) is compared with the gRNA sequence of the whole CRISPR genome to identify the gene enriched in the zinc transport differential intestinal epithelial cell double gene knockout cell library with weak zinc transport capacity. The gene is the target amino acid chelate zinc metabolism gene.

[0006] Preferably, the single gene in step (1) is ZIP4 or ZIP5.

[0007] Preferably, the intestinal epithelial cells in step (1) are colon cancer epithelial cells CaCO2 or porcine jejunal epithelial cells IPEC-J2.

[0008] Preferably, step (1) involves designing gRNA for genes related to zinc transport function, constructing a knockout plasmid containing the CRISPR-Cas9 system and gRNA using the designed gRNA, and simultaneously transfecting the constructed knockout plasmid and lentiviral packaging plasmid into 293T cells. After 3-4 days, the lentiviral supernatant is collected, and the collected lentiviral supernatant is added to the target cells containing genes related to zinc transport function. The target cells are cultured and then screened with blastomycin to obtain a stable Cas9-expressing target gene knockout cell line.

[0009] Preferably, step (2) involves packaging the human whole genome knockout library plasmid with lentivirus and then transfecting it into a single-gene knockout cell library. After culturing, the cell library is screened with puromycin to obtain a stable double-gene knockout cell library expressing Cas9.

[0010] Preferably, the zinc source in step (3) is amino acid chelated zinc with a zinc concentration of 100 μM and a zinc source treatment time of 6 h.

[0011] Preferably, the fluorescent dye in step (3) is Zinpy-1 dye, and the staining time is 30 min.

[0012] Preferably, the flow cytometer sorting in step (3) is performed at least three times.

[0013] Preferably, the targeted amino acid chelating zinc metabolism genes mentioned in step (5) are CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, and ZNF570.

[0014] The aforementioned targeted amino acid chelating zinc metabolism genes can be used to design new trace element feed additives, prepare feed additives to improve animal nutritional status, and also to prepare nutritional supplements to improve human zinc nutritional status or drugs to treat human zinc metabolism-related diseases.

[0015] The present invention also provides a Zn metabolism regulatory gene library, wherein the genes in the Zn metabolism regulatory gene library are CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, and ZNF570.

[0016] The present invention will be further described below: The core of this invention lies in constructing a "clean background" screening model. First, a genome-wide knockout library is introduced into a cell line with a key zinc transport gene (such as ZIP4) knocked out, thereby amplifying the signals of other genes' roles in amino acid chelate zinc metabolism. Intracellular zinc levels are labeled using a zinc source and a zinc ion fluorescent dye (such as Zinpyr-1), and the cell library with the weakest zinc uptake capacity is sorted by flow cytometry. Finally, gRNA sequencing and bioinformatics analysis of this cell library identify enriched genes. The knockout of these genes leads to decreased zinc uptake capacity; therefore, they are considered key target genes regulating amino acid chelate zinc metabolism.

[0017] In summary, this invention establishes a method for high-throughput screening of amino acid chelation zinc-specific metabolic genes, and also obtains a library of Zn metabolism regulatory genes. Attached Figure Description

[0018] Figure 1Figure A shows the transport characteristics of zinc from different zinc sources in the zinc transporter gene knockout model; Figure B shows the sequencing results of the ZIP4, ZIP5, DMT1 and ZIP14 gene knockouts; Figure C shows the intracellular uptake level of zinc sulfate in the zinc transporter gene knockout model; Figure D shows the transport level of zinc sulfate from the intracellular to the basal layer in the zinc transporter gene knockout model. Figure 2 This study aimed to sort and validate the epigenetic characteristics of Caco-2 cell lines with differential zinc transport. Figure A shows the flow cytometry sorting of Caco-2 cell lines with differential zinc transport using a double gene knockout technique, and Figure B shows the epigenetic validation of these cell lines. R3 represents cell lines with weak intracellular fluorescence intensity, indicating weak zinc transport capacity, while R4 represents cell lines with strong intracellular fluorescence intensity, indicating strong zinc transport capacity. Zip4- / / - represents double gene knockout cell lines based on ZIP4 knockout, and different superscript letters indicate significant differences. P <0.05. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of the embodiments of the present invention clearer, further descriptions will be provided below in conjunction with specific implementations of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Other implementation methods obtained by those skilled in the art based on the embodiments disclosed in the present invention without creative effort should all fall within the scope of protection of the present invention. Materials not specifically described in the embodiments (such as primers, gRNA, human whole-genome knockout library plasmids, lentiviral materials, etc.) are all conventionally known designs in the art or commercially available materials provided by commercial companies.

[0020] Example 1 1. Zn 2+ Construction of transport-deficient cell lines gRNAs encoding the zinc transporter genes ZIP4, ZIP5, DMT1, and ZIP14 of Caco-2 were designed (gRNA design was performed at the following website: https: / / www.benchling.com / crispr / , using randomized design principles, and is known to those skilled in the art). Knockout plasmids containing the Crispr-Cas9 system and gRNAs were constructed using the designed gRNAs. When 293T cell lines reached 70% confluence, the constructed knockout plasmids and commercially available lentiviral packaging plasmids were simultaneously transfected into 293T cells. Lentiviral supernatant was collected 3-4 days later, and the collected lentivirus was added to Caco-2 target cells containing the zinc transporter-related genes ZIP4, ZIP5, DMT1, or ZIP14, respectively. The medium was changed 16 h after transfection. After selection with blastidin, stable Caco-2 single-gene knockout cell lines expressing Cas9 were obtained, and sequencing was performed to verify whether the target genes were knocked out or their off-target sites.

[0021] 2. Lentiviral transfection of Crispr whole genome and gene-deficient cell lines Human whole genome knockout library plasmids (designed and synthesized according to random principles, known to those skilled in the art, or commercially available from third-party companies) were packaged with lentivirus (commercial lentivirus) and transfected into Caco-2 single-gene knockout cell lines. After 16 h of transfection, the medium was changed, and after selection with puromycin (Puro), Caco-2 double-gene knockout cell lines stably expressing Cas9 were obtained.

[0022] 3. Flow cytometry-based screening Caco-2 double-gene knockout cell lines were seeded in T75 cell culture flasks until 70-80% confluence. 100 μM zinc sulfate, zinc hydroxymethionine, and zinc glycine were added to the Caco-2 double-gene knockout cells, respectively. After 6 h of treatment, the cell supernatant was discarded, and the cells were washed twice with PBS. The Caco-2 double-gene knockout cells were stained with Zinpy-1 dye for 30 min, washed twice with PBS, and the cells were collected with trypsin. After centrifugation at 500×g for 5 min, the supernatant was discarded. The cells were resuspended in basal medium to obtain a single-cell suspension. Flow cytometry was used to screen Caco-2 double-gene knockout cell libraries with different zinc source transport differences based on intracellular zinc concentration.

[0023] 4. Phenotypic identification of the selected cell set Caco-2 cells in the logarithmic growth phase with double gene knockout were seeded into 6-well plates and cultured for 24–48 h. The culture medium was discarded, and the cells were washed twice with 1 mL of HBSS. 1 mL of 25 μM Zinpyr-1 dissolved in HBSS was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 30 min. The dye was discarded, and each well was washed three times with 1 mL of HBSS. 1 mL of HBSS was added to each well. Fluorescence images were captured using a fluorescence microscope (Leica, DMI3000B) at an excitation wavelength of 495 nm.

[0024] 5. gRNA sequencing analysis and gene annotation Whole genome extraction was performed on the Caco-2 double gene knockout cell line using a Cell Genome Extraction Kit (TransGen, Beijing) (methods as described in the kit instructions). The genome was submitted to a sequencing company for sequencing to obtain the gRNA sequences of the Caco-2 double gene knockout cell line. The obtained gRNA sequences were compared with the gRNA sequences of the CRISPR whole genome to identify genes enriched in cells with weak zinc transport capacity in the zinc transport differential Caco-2 double gene knockout cell line (using conventional existing techniques).

[0025] Example 2 Following the method in Example 1, a Caco-2 cell line with ZIP4 gene knockout was constructed, and then a human whole-genome knockout library was added. After screening with puromycin, a dual-gene knockout cell line based on ZIP4 knockout was obtained. Different zinc sources were added to this cell line for zinc treatment, and cell libraries with different zinc transport capabilities were sorted by flow cytometry. Figure 2 A), and cell libraries with low and high intracellular zinc levels were collected separately. To obtain cell libraries with weak zinc transport capacity and stable phenotypes, the cell line was subjected to flow cytometry sorting three times, and cell lines with different zinc transport capacity obtained by flow cytometry screening were purified and amplified. The zinc transport phenotype of the cell lines was verified, and the results are as follows. Figure 2 As shown in B, the purified dual-gene knockout cell lines exhibited different uptake abilities of zinc sulfate, zinc hydroxymethionine, and zinc glycine, with significant differences (P < 0.05).

[0026] High-throughput sequencing was performed on the phenotypically stable cell line with weak zinc transport capacity (Zip4- / / -R3). The gRNA in Zip4- / / -R3 cells was compared with the gRNA in the whole genome library. The results are shown in Table 1. The cell line with weak zinc transport capacity obtained based on three flow cytometry sorting was enriched with the following 11 genes: CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, and CST6. These genes constitute a Zn metabolism regulatory gene library.

[0027] Table 1. Percentage of zinc transport-specific genes screened based on genome-wide knockout libraries (%)

[0028] Note: ZnS1, ZnS2, and ZnS3: three replicates from high-throughput sequencing of the zinc sulfate group; ZnM1, ZnMM2, and ZnMM3: three replicates from high-throughput sequencing of the zinc hydroxymethionine group; ZnS1, ZnS2, and ZnS3: three replicates from high-throughput sequencing of the zinc glycine chelate group.

Claims

1. A screening method based on a CRISPR whole-genome library targeting amino acid chelation zinc metabolism genes. Its characteristic is that... The screening method includes the following steps: (1) Construct a single-gene knockout cell line of intestinal epithelial cells that stably expresses Cas9 and has zinc transport function defects by using CRISPR-Cas9 technology; the single gene is ZIP4, ZIP5 or ZIP14, which are related to zinc transport function. (2) The Crispr whole genome knockout library plasmid was packaged with lentivirus and then transfected into the intestinal epithelial cell gene knockout cell line constructed in step (1) to obtain an intestinal epithelial cell double gene knockout cell library that stably expresses Cas9. (3) Treat the intestinal epithelial cell double gene knockout cell library constructed in step (2) with at least one zinc source, and then stain the intestinal epithelial cell double gene knockout cell line in the intestinal epithelial cell double gene knockout cell library with zinc ion sensitive fluorescent dye. Then sort the intestinal epithelial cell double gene knockout cell library with different zinc transport intracellular zinc concentration by flow cytometry. (4) Extract whole genome DNA from the zinc transport differential intestinal epithelial cell double gene knockout cell library sorted in step (3), and amplify the gRNA sequence of the zinc transport differential intestinal epithelial cell double gene knockout cell line using the whole genome DNA as a template. (5) By bioinformatics analysis, the gRNA sequence obtained in step (4) is compared with the gRNA sequence of the whole CRISPR genome to identify the gene enriched in the zinc transport capacity of the zinc transport differential intestinal epithelial cell double gene knockout cell line. The gene is the target amino acid chelate zinc metabolism gene.

2. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, The single gene mentioned in step (1) is ZIP4 or ZIP5.

3. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, The intestinal epithelial cells mentioned in step (1) are colon cancer epithelial cells CaCO2 or porcine jejunal epithelial cells IPEC-J2.

4. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, Step (1) involves designing gRNAs for genes related to zinc transport function. Using the designed gRNAs, a knockout plasmid containing the CRISPR-Cas9 system and gRNAs is constructed. The constructed knockout plasmids and commercially available lentiviral packaging plasmids are simultaneously transfected into 293T cells. After 3-4 days, the lentiviral supernatant is collected, and the collected lentivirus is added to the target cells containing genes related to zinc transport function. The target cells are cultured and then screened with blastomycin to obtain a stable Cas9-expressing target gene knockout cell line.

5. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, Step (2) involves packaging the human whole genome knockout library plasmid with lentivirus and transfecting it into single-gene knockout cell lines. After culturing, the cells are screened with puromycin to obtain a stable double-gene knockout cell library expressing Cas9.

6. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, The zinc source in step (3) is one or more amino acid chelated zinc, the zinc concentration is 100 μM, and the zinc source treatment time is 6 h; the fluorescent dye is Zinpy-1 dye, and the staining time is 30 min; the flow cytometry sorting is performed at least three times.

7. The screening method for targeting amino acid chelation zinc metabolism genes based on a CRISPR whole-genome library as described in claim 1, characterized in that, The targeted amino acid chelating zinc metabolism genes mentioned in step (5) are CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, and ZNF570.

8. The application of the targeted amino acid chelation zinc metabolism gene as described in claim 7 in the targeted design of new trace element feed additives.

9. The application of the targeted amino acid chelating zinc metabolism gene as described in claim 7 in the preparation of feed additives to improve animal nutritional status, nutritional supplements to improve human zinc nutritional status, or drugs to treat human zinc metabolism-related diseases.

10. A Zn metabolism regulatory gene library, characterized in that, The genes in the Zn metabolism regulatory gene library are CST6, LYPD4, SMIM6, SLC50A1, PCDH8, SRPK1, HGFAC, KRTAP10-5, GNAT1, and ZNF570.

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