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KR-102963498-B1 - Method for generation of hypoallergenic cat using CRISPR/Cas9 system

KR102963498B1KR 102963498 B1KR102963498 B1KR 102963498B1KR-102963498-B1

Abstract

The present invention relates to a method for producing a cat with low secretion of allergy-inducing proteins using a CRISPR/Cas9 system and a method for cloning a cat with low secretion of allergy-inducing proteins using cytoplasm injection clone technology. The present invention developed a high-efficiency sgRNA for Fel d 1 gene knockout and established an efficient transgenic cat production technology using cytoplasmic microinjection. Accordingly, transgenic cats produced according to the method of the present invention suppress the secretion of Fel d 1 protein, an indoor allergen (Felis Domesticus Allergen), thereby having the effect of lowering the frequency of cat allergy in humans, and can be usefully utilized in the pet market.

Inventors

  • 공일근

Assignees

  • 경상국립대학교산학협력단

Dates

Publication Date
20260512
Application Date
20230223

Claims (16)

  1. (a) A step of culturing oocytes collected from a donor female cat in an in vitro maturation (IVM) medium; (b) A step of producing a cat embryo by in vitro fertilization (IVF) of the mature oocyte from step (a) and sperm collected from a male cat; (c) transfecting a mixture of sgRNA that specifically binds to the CH1 gene of Fel d 1 protein, sgRNA that specifically binds to the CH2 gene of Fel d 1 protein, and Cas9 mRNA into the cytoplasm of the cat embryo from step (b); (d) a step of culturing the transformed embryo from step (c) above in an in vitro culture (IVC) medium to develop it to a blastocystis; (e) a step of producing a first-generation transgenic cat by implanting the blastocyst from step (d) into the fallopian tube of a recipient female cat; (f) a step of producing a second generation of transgenic cats by selecting transgenic cats having the CH2 Mosaic genotype and transgenic cats having the CH2 +/- genotype for the CH2 gene of Fel d 1 protein from among the first generation transgenic cats of step (e) above, and then crossing them; and (g) a step of analyzing the genotype of the second-generation transgenic cat from step (f) above to select homozygous mutants for the Fel d 1 protein; comprising a method for producing a Fel d 1 gene knockout transgenic cat, The sgRNA that specifically binds to the CH1 gene of the Fel d 1 protein in step (c) above is represented by the nucleotide sequence of SEQ ID NO. 1, and The sgRNA that specifically binds to the CH2 gene of the Fel d 1 protein in step (c) above is represented by the nucleotide sequence of SEQ ID NO. 2, and A method characterized in that the above homozygous mutant has a CH2 -/- genotype for the CH2 gene of Fel d 1 protein.
  2. In paragraph 1, A method characterized in that the culture of step (a) above is performed at 30 to 45°C for 3 to 5 hours.
  3. In paragraph 1, A method characterized in that the in vitro fertilization of step (b) above is performed at 30 to 45°C for 5 to 7 hours.
  4. delete
  5. delete
  6. In paragraph 1, A method characterized in that the embryo at step (c) above is a zygote at the single-cell stage.
  7. In paragraph 1, A method characterized in that the transfection of step (c) above is performed by a microinjection method.
  8. In paragraph 1, A method characterized in that the culture of step (d) above is performed at 30 to 45°C for 5 to 7 hours.
  9. delete
  10. delete
  11. A cat with low secretion of allergenic protein produced by the method of paragraph 1.
  12. In Paragraph 11, A cat with low secretion of allergy-causing proteins, characterized in that the above-mentioned allergy-causing protein is an indoor allergen.
  13. In Paragraph 12, A cat with low secretion of allergy-inducing proteins, characterized in that the above indoor allergen is Fel d 1 protein.
  14. A step of producing cloned embryos using cytoplasm injection clone technology from somatic cells isolated from cats with low secretion of the allergenic protein of claim 11; and A method for producing a Fel d 1 gene knockout transgenic cloned cat comprising the step of implanting the cloned embryo into the fallopian tube of a female cat recipient.
  15. (a) A step of culturing oocytes collected from a donor female cat in an in vitro maturation (IVM) medium; (b) A step of producing a cat embryo by in vitro fertilization (IVF) of the mature oocyte from step (a) and sperm collected from a male cat; (c) transfecting a mixture of sgRNA that specifically binds to the CH1 gene of Fel d 1 protein, sgRNA that specifically binds to the CH2 gene of Fel d 1 protein, and Cas9 mRNA into the cytoplasm of the cat embryo from step (b); (d) a step of culturing the transformed embryo from step (c) above in an in vitro culture (IVC) medium to develop it to a blastocystis; (e) a step of producing a first-generation transgenic cat by implanting the blastocyst from step (d) into the fallopian tube of a recipient female cat; (f) a step of producing a cloned embryo using cytoplasm injection clone technology from somatic cells isolated from the first-generation transgenic cat; and (g) a step of implanting the cloned embryo into the fallopian tube of a female cat; a method for producing a Fel d 1 gene knockout transgenic cloned cat.
  16. Allergenic protein-low secretion cat produced by the method of Paragraph 15.

Description

Method for generation of hypoallergenic cat using CRISPR/Cas9 system The present invention relates to a method for producing a cat with low allergy-causing protein secretion using a CRISPR/Cas9 system and a method for cloning a cat with low allergy-causing protein secretion using cytoplasm injection clone technology. In general, cats (Felis Catus; Fel d) are very important and useful animals to humans as disease model animals as well as companion animals. Meanwhile, cats produce eight types of Felis Domesticus Allergens, namely Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 5, Fel d 6, Fel d 7, and Fel d 8, which cause allergic conjunctivitis, allergic rhinitis, and life-threatening atopic asthma in humans sensitive to feline allergens (T. Virtanen et al., Mammalian Allergens, Allergens and Allergen Immunotherapy: Subcutaneous, Sublingual and Oral (fifth edition), 217-233 (2014)). In particular, IgE-mediated sensitization to the major allergen Fel d 1 affects about 10% of the Western world population (S. K. Chan, D. Y. M. Leung, Allergy Asthma Immunol. Res. 10(2), 97-105 (2018)). Fel d 1 causes 88 to 95% of allergic reactions in people sensitive to cat allergens, inducing the most severe allergic reactions in humans, and is produced in the sebaceous glands of the hair and salivary glands. High concentrations of Fel d 1 are transferred when skin (pelt) and hair are groomed. Due to its small size, Fel d 1 is transferred from cat fur into the indoor environment via the air, saliva, and dander, causing sensitization in sensitive individuals, and can adhere to surfaces such as clothing, carpets, and furniture. Compared to mice and rats, which are the most commonly used animal models, cats have several unique advantages, including a longer lifespan and high similarity to certain human tissues. Therefore, many research groups have made great efforts to genetically modify cats, but there is a problem in producing transgenic cats due to the limitations of the feline reproductive mechanism resulting from the limited number of eggs and embryos. Meanwhile, since CRISPR was discovered in E. coli by Japanese scientist Yoshizumi Ishino in 1987, technology has been developed to produce various transgenic animals in new ways using the CRISPR/Cas9 system, which is created by combining CRISPR with Cas9, a nuclease that cuts DNA. The CRISPR (clustered, regular interspaced, short palindromic repeats)/Cas9 (CRISPR-associated 9) system in bacteria and archaea was first discovered in 1987 as an ‘immune system’ related to the degradation of external plasmids and viruses. Since then, the system has been widely used as a highly efficient genome editing technology capable of various applications. This is an endonuclease splicing technology commonly referred to as gene scissors, which has the advantage of allowing for more efficient and easier gene manipulation than first-generation Zinc-Finger Nucleases (ZFNs) and second-generation Transcription Activator-like Effector Nucleases (TALENs). This CRISPR/Cas9 system operates on the principle of neutralizing gene function by attacking the Cas9 target site within a specific gene through the induction of a single guide RNA (sgRNA) identical to the target site within that gene, thereby causing gene insertion and loss. If it is possible to artificially induce insertion, loss, or modification in genes located in specific parts of a chromosome in this way, it could be widely utilized not only in genetics and molecular biology research but also in the study of diseases related to specific genes. However, the CRISPR/Cas9 system has so far only been used by direct microinjection into the embryos of a limited number of livestock and rodent animals. Accordingly, the inventors developed an sgRNA having a high knockout efficiency for the CH2 gene of the Fel d 1 protein, and produced a transgenic cat with the CH2 gene knocked out by microinjecting a CRISPR/Cas9 system containing the sgRNA into the cytoplasm of a feline zygote. In addition, to maintain the lineage of the transgenic cat, they produced a transgenic cloned cat using cytoplasmic cloning technology (CICT) and completed the present invention. Figure 1 is a figure showing the positions of sgRNA candidates in the CH1 and CH2 genes of the Fel d 1 protein in one embodiment of the present invention. FIG. 2 is a figure showing the results of in vitro validation of an sgRNA candidate targeting the CH1 gene in one embodiment of the present invention. FIG. 3 is a figure showing the results of in vitro validation of an sgRNA candidate targeting the CH2 gene in one embodiment of the present invention. FIG. 4 is a flowchart briefly illustrating the process of producing a gene-edited cat for the Fel d 1 protein in one embodiment of the present invention. Figure 5 is a figure showing the results of producing a first-generation transgenic cat in which the Fel d 1 protein CH2 gene was knocked out by a CRISPR/Cas9 system in one embodiment of the present invention.