期刊
GENOME RESEARCH
卷 31, 期 5, 页码 -出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1101/gr.271312.120
关键词
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资金
- Transgenic Research Grants [2016ZX08009003]
- National Natural Science Foundation of China [32002180]
- 2020 Research Program of Sanya Yazhou Bay Science and Technology City [202002011]
The authors developed an insertional mutagenesis strategy (HIT-trapping) and successfully created mutant alleles for 21 disease-related genes in porcine fibroblasts, achieving a significant improvement over previous methods.
Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.
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