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Background <p>Gene targeting via CRISPR/Cas9 has become a powerful tool to create animal models for human genetic disorders. Although CRISPR/Cas9 mediated gene editing is relatively simple, factors such as mouse strain, breeding performance, and quality and recovery of the embryos, can limit the overall outcome. Genetic manipulations are commonly carried out using embryos from a well characterized C57BL6 strain of mouse however, the ability for direct gene editing in strains such as non-obese diabetic (NOD) mice, which is an important strain to create mouse models for autoimmune type 1 diabetes (TD1), immunology, cancer and infectious diseases, is quite limiting. Therefore, gene modification in NOD mice in one way is carried out via a time-consuming cross breeding with the transgenic animals created in a more permissive C57BL6 strain for several generations which can take long time and consume valuable resources. Direct gene manipulation in the NOD mouse background will greatly expedite the creation of genetically engineered mouse models bypassing the complicated steps of backcrossing thus saving almost two years and valuable resources and help promoting 3 R principles of refinement of animal welfare.</p> Results <p>We present improved and streamlined conditions for gene manipulation directly on the pure NOD background using embryos from NODShiLtJ mouse, a strain commonly used for studies on autoimmune TD1 and animal models for potential applications in cancer, immunology, and infectious disease research. High efficiency, 80–90% success rate, was achieved via CRISPR/Cas9 genome editing in pups born from embryos recovered from naturally mated 4-week-old NODShiLtJ females superovulated under specific time schedules.</p> Conclusions <p>We demonstrate that 4-week-old NODShiLtJ females superovulated and mated under specific time requirements can produce healthy and robust embryos for gene manipulation saving significant resources and maintenance costs that will help in 3R principles of refinement to improve animal welfare. Similar conditions may be applied to create new genetic modifications in complex NOD/SCID Gamma (NSG) and NOD/SCID Rag (NRG) mice for dissecting mechanisms of immunology as well as generation of mouse models for infectious diseases for underlying conditions in human patients.</p>

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A streamlined approach for gene editing in non-obese diabetes (NOD) mice via CRISPR/Cas9

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月26日

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  • Kyoungin Cho,
  • Michail S. Lionakis,
  • Jaspal S. Khillan

摘要

Background

Gene targeting via CRISPR/Cas9 has become a powerful tool to create animal models for human genetic disorders. Although CRISPR/Cas9 mediated gene editing is relatively simple, factors such as mouse strain, breeding performance, and quality and recovery of the embryos, can limit the overall outcome. Genetic manipulations are commonly carried out using embryos from a well characterized C57BL6 strain of mouse however, the ability for direct gene editing in strains such as non-obese diabetic (NOD) mice, which is an important strain to create mouse models for autoimmune type 1 diabetes (TD1), immunology, cancer and infectious diseases, is quite limiting. Therefore, gene modification in NOD mice in one way is carried out via a time-consuming cross breeding with the transgenic animals created in a more permissive C57BL6 strain for several generations which can take long time and consume valuable resources. Direct gene manipulation in the NOD mouse background will greatly expedite the creation of genetically engineered mouse models bypassing the complicated steps of backcrossing thus saving almost two years and valuable resources and help promoting 3 R principles of refinement of animal welfare.

Results

We present improved and streamlined conditions for gene manipulation directly on the pure NOD background using embryos from NODShiLtJ mouse, a strain commonly used for studies on autoimmune TD1 and animal models for potential applications in cancer, immunology, and infectious disease research. High efficiency, 80–90% success rate, was achieved via CRISPR/Cas9 genome editing in pups born from embryos recovered from naturally mated 4-week-old NODShiLtJ females superovulated under specific time schedules.

Conclusions

We demonstrate that 4-week-old NODShiLtJ females superovulated and mated under specific time requirements can produce healthy and robust embryos for gene manipulation saving significant resources and maintenance costs that will help in 3R principles of refinement to improve animal welfare. Similar conditions may be applied to create new genetic modifications in complex NOD/SCID Gamma (NSG) and NOD/SCID Rag (NRG) mice for dissecting mechanisms of immunology as well as generation of mouse models for infectious diseases for underlying conditions in human patients.