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

By constructing intestinal epithelial cell lines with zinc transport dysfunction, and combining CRISPR-Cas9 technology and flow cytometry, amino acid chelating zinc metabolism genes were screened out. This overcame the shortcomings of existing screening methods and enabled efficient screening and application in the development of products related to improving zinc metabolism.

CN122012489APending Publication Date: 2026-05-12INSTITUTE OF SUBTROPICAL AGRICULTURE CHINESE ACADEMY OF SCIENCES
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Patent Information

Application Number
CN202511905371.9
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-12-17
Publication Date
2026-05-12

AI Technical Summary

Technical Problem

Existing technologies lack efficient and specific methods for screening amino acid chelate zinc metabolism genes, which limits the in-depth research and development of related technologies and the promotion and application of products.

Method used

A single-gene knockout cell line of intestinal epithelial cells with zinc transport dysfunction was constructed. A whole-genome knockout library was constructed using CRISPR-Cas9 technology. Combined with zinc source treatment and fluorescent dye labeling, cells with differential zinc transport were sorted by flow cytometry. Whole-genome DNA was extracted and bioinformatics analysis was performed to identify genes for amino acid chelation zinc metabolism.

Benefits of technology

We have achieved high-throughput screening of amino acid chelation zinc-specific metabolic genes, obtained a Zn metabolism regulatory gene library, and developed feed additives and nutritional supplements for improving zinc nutrition status in animals and humans, as well as drugs for treating zinc metabolism-related diseases.

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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.
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