Gene function & annotation

Construction of a Gene?Perturbation Atlas Model in Chicken via the CRISPRà¢â‚¬â€œCas9 System

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Abstract

IntroductionGene editing offers a precise method for animal genetic improvement, shifting breeding from random selection to targeted optimization. In poultry, traits like growth rate and feed efficiency impact both economics and public health. However, many of these traits are controlled by genes with unknown functions. Traditional gene-editing methods face challenges such as low throughput and cellular interference. To overcome these, we developed the Inducible Gene-Perturbation Model of Chicken (IGPMC), a transgenic model enabling simultaneous expression of multiple sgRNAs, allowing efficient gene knockout and the creation of chimeric chickens. This system facilitates rapid gene function evaluation and accelerates genetic improvement.Materials and MethodsBased on the research accumulated and multi-omics data analysis, we selected 31 key genes related to growth traits. Using the PiggyBac vector, we introduced an sgRNA library into primordial germ cells and achieved controlled expression of gRNAs through the Cre-LoxP system and drug induction. After obtaining stable PGC cell lines, we directly microinjected them into recipient embryos and cultured them in vitro, yielding chickens that expressed the gene-editing elements. Drug induction further generated chimeric chickens in which each cell exhibited different editing effects. High-throughput sequencing was used to track dynamic changes in sgRNAs. Combined with phenotypic and transcriptomic data, we established a mapping relationship between gene perturbations and phenotypes to create the chicken functional gene perturbation atlas.ResultsWe have completed the construction of all elements and the system setup for the perturbation map, including: 1) successfully optimizing the isolation, culture, and efficient transfection methods for primordial germ cells, and constructing a PGC cell line; 2) designing sgRNAs for the 31 selected genes, along with the construction of expression vectors and the identification of highly efficient candidates via DF-1 cell screening, culminating in tandem vector assemblies; 3) successfully verifying the single recombination property of the Cre-LoxP system in DF1 cells, ensuring that each cell expresses only a single sgRNA after induction. As a proof of concept, we constructed a tandem vector targeting five genes and conducted a preliminary functional assessment in DF-1 cells.Conclusion and DiscussionThis study aims to build a high-throughput gene research platform based on CRISPR-Cas9 gene editing technology and the Cre-LoxP regulated expression system to map the functional gene perturbation atlas in chickens. IGPMC has significant advantages over traditional virus-based strategies. Furthermore, IGPMC allows for the rapid generation of single-gene edited chicken, which can be used to verify and further explore candidate genes identified in preliminary studies. Currently, the candidate genes designed for IGPMC focus primarily on growth traits, and future research will expand to study the functions of other types of genes. In conclusion, IGPMC offers an efficient technical pathway and data support for high-throughput poultry functional gene research and breed improvement.

Keywords: 2026

How to Cite:

Guo, X., Wu, H., Wang, Y., Li, G. & Hu, X., (2026) “Construction of a Gene?Perturbation Atlas Model in Chicken via the CRISPRà¢â‚¬â€œCas9 System”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2285438. doi: https://doi.org/10.31274/wcgalp.23708

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