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A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products

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Abstract

Sex selection technologies have immensely impacted swine production globally. Conventional earlier embryo sex identification methods require professional technicians and sophisticated laboratory instruments. Rapid on-site gender identification of porcine embryos and pork products remains challenging. In this study, we developed a CRISPR/Cas12a-based fluorescence visualization point-of-care sex determination test that is rapid, accurate and easy to implement on-site. The CRISPR/Cas12a assay coupled with either the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) employs precisely designed primers and single-guide RNAs targeting the sex-determining region Y (SRY) and the zinc finger protein X-linked (ZFX) genes. PCR and LAMP amplicons were cleaved with the subsequent generation of fluorescing products detectable with portable blue and ultraviolet light transilluminators. Approximately two copies per microliter of the ZFX and SRY genes were detected using the RApid VIsual CRISPR (RAVI-CRISPR) assay. This method is a sensitive, inexpensive, versatile, and point-of-care test. The technology has other potential applications like determining the sex of diverse livestock species, detecting livestock disease-causing pathogens and evaluating the quality of meat products.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (32072685), the Fund of Modern Industrial Technology System of Pig (CARS-35), and the Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province (KLAEMB-2020-05).

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Author notes

  1. Dagang Tao, Jiajia Liu, Qiushi Li have contributed equally to the study.

Authors and Affiliations

  1. Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Dagang Tao, Bingrong Xu, Jing Xu, Yunlong Ma, Jinxue Ruan & Shengsong Xie

  2. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, 611130, People’s Republic of China

    Jiajia Liu & Lili Niu

  3. College of Biological Engineering and Food Science, Hubei University of Technology, Wuhan, 430070, People’s Republic of China

    Qiushi Li

  4. Yangshan Customs, Shanghai, 201306, People’s Republic of China

    Yu Jiang

  5. International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi, 00100, Kenya

    Emmanuel Mulaya Khazalwa

  6. Institute of Animal Husbandry and Veterinary, Wuhan Academy of Agricultural Science, Wuhan, 430208, People’s Republic of China

    Ping Gong

  7. Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Wuhan, 430070, People’s Republic of China

    Jinxue Ruan & Shengsong Xie

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  1. Dagang Tao
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  2. Jiajia Liu
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  11. Lili Niu
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  12. Shengsong Xie
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Contributions

SSX, DGT, and JJL: conceptualization. JJL and DGT: Methodology software. JJL and YJ: validation. QSL, PG, and JX: formal analysis. EMK: investigation. DGT, JJL and QSL: resources. SSX and LLN: data curation. JXR and YLM: writing—original draft preparation. SSX, JJL, DGT and QSL: writing—review and editing, SSX, DGT and EMK: visualization. DGT, BRX and QSL: supervision and project administration. SSX and LLN: funding acquisition. SSX All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Lili Niu or Shengsong Xie.

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Tao, D., Liu, J., Li, Q. et al. A Simple, Affordable, and Rapid Visual CRISPR-Based Field Test for Sex Determination of Earlier Porcine Embryos and Pork Products. Mol Biotechnol 65, 263–272 (2023). https://doi.org/10.1007/s12033-022-00532-x

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