A new gene-targeted integration strategy, Tild-CRISPR.

Recently, Yang Hui Research Group of the Center for Excellence in Brain Science and Intelligent Technology/Institute of Neuroscience of the Chinese Academy of Sciences, in collaboration with Chen Zijiang, a professor at Shandong University's affiliated reproductive hospital and Renji Hospital affiliated with Shanghai Jiaotong University Medical School, designed a new gene-targeted integration strategy, Tild-CRISPR, which can achieve efficient and accurate gene knock-in in mouse and human cells, not only for the efficient construction of mouse animal models, but also for the study of gene function and potential gene therapy.
research by Tild-CRISPR Allows for Precise and Gene Knockin in Mouse and Human Cells was published on May 21 in DevelopmentAl Cell.
CRISPR/Cas9-mediated gene editing method greatly promotes the integration of genetically modified targets in the body and in vitro.
traditional isologoitation (HR)-mediated integration strategy is inefficient in animal embryos and tissues.
non-ho-source end join (NHEJ) and microhologoite arm-mediated end joining (MMEJ) strategies can only increase efficiency in certain systems.
previous research by the research team, the same arm-mediated end joint (HMEJ) strategy can achieve efficient targeted integration in mouse embryos as well as in mice.
, there are still technical barriers to efficiently constructing conditional or large fragment genes into mouse models, and achieving efficient targeted integration in human embryos.
the study, based on the CRISPR/Cas9 system, designed a new targeted integration strategy, Tild-CRISPR (Targeted Integration with Linearized dsDNA-CRISPR), in which a genetically modified donor containing 800bp homologous arm was injected into a fertilized egg with Cas9 mRNA and a single-guideRNA through PCR amplification or precise enzymatic cutting.
compared to the existing targeted strategies, the method showed the highest knock-in efficiency in injected mouse embryos, and successfully obtained knock-in mice from 0.8kb to 6.0kb of different lengths in 6 different sites.
compared to HR or HMEJ-mediated methods, Tild-CRISPR showed more powerful in vivo DNA knock-in capability in intrauterine electrotransfer synths.
more importantly, in human embryos, the Tild-CRISPR method is 12 times more efficient than the HR-mediated method, which greatly facilitates research on human embryo development and the repair of disease mutations.
the study established a new gene-targeted integration strategy Tild-CRISPR, through in vitro PCR amplification or precision enzyme cutting to obtain a linear genetically modified donor containing 800bp homologous arm, can achieve efficient DNA targeting integration in mouse and human cells, for the study of genetic function in the body and the development of potential gene therapy provides a new way of thinking.
the study was conducted by Yao Wei, assistant researcher at the Institute of Neurology, Wang Xing, ph.d., and Zhang Meiling, Ph.D. of The Center for Reproductive Medicine at Renji Hospital, under the guidance of Yang Hui and Chen Zijiang. Meng Feilong, a researcher at the Institute of Biochemistry and Cell Biology of the Shanghai Institute of Life Sciences of the Chinese Academy of Sciences,
, as well as the experimental animal platform, molecular cell platform and optical imaging platform of the Institute of Neurology, contributed to this research.
the research has been supported by the National Science and Technology Commission, the Shanghai Science and Technology Commission, the National Natural Science Foundation of China, and the Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics.
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