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Efficient and Precise Gene Knock-in Achieved in Mouse and Human Cells Using Tild-CRISPR

May 22, 2018     Email"> PrintText Size

CRISPR/Cas9-mediated genome editing has greatly facilitated targeted integration of transgenes both in vitro and in vivo, while, the more generally used homologous recombination (HR) is inefficient for achieving gene integration in animal embryos and tissues, although non-homologous end-joining (NHEJ)- or microhomology- mediated end-joining (MMEJ)-based methods can elevate the efficiency in some systems.

Previous studies of YANG Hui’s Lab at Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences showed that in vivo cleavage of transgene donors with ~800 bp of homology arms could promote CRISPR-mediated targeted integration in animal embryos and tissues in vivo. However, technical barriers still exist in efficiently creating mice with conditional alleles or large insertion, and precise knock-in in human embryos.

In a study published in Developmental Cell on May 21, 2018, Dr. YANG’s Lab, in collaboration with Dr. CHEN Zijiang's group at the Shandong Provincial Hospital affiliated to Shandong University, RenJi Hospital Shanghai Jiao Tong University School of Medicine, demonstrated that the efficient and precisely targeted integration can be achieved by Tild-CRISPR in mouse and human cells.

Based on CRISPR/Cas9 system, the researchers designed a new targeting method termed as Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes.

Tild-CRISPR exhibited the highest knock-in efficiency compared with all other targeting strategies with various types of transgene donor in mouse embryos. Various insertions, from 0.8 to 6.0 kb, could be precisely integrated into different loci. Compared with HR- or HMEJ-based methods, Tild-CRISPR showed robust DNA knock-in using in utero electroporation in the mouse brain.

Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knock-in efficiency than HR-based methods in human embryos, making it suitable for studying human embryo development and correcting pathologic gene mutation.

This work devised a new method called Tild-CRISPR, a targeting strategy using PCR amplified or precisely enzyme-cut transgene donor, yields knock-in efficiency in mouse and human embryos, as well as the mouse brain in vivo. With higher knock-in efficiency in mouse and human cells, Tild-CRISPR holds a great promise for applications such as studying gene functions in vivo and developing potential gene therapies.

 

Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy using PCR amplified or precisely enzyme-cut transgene donor, yields robust knock-in efficiency in mouse and human embryos, as well as mouse brain in vivo. (Image by YAO Xuan) 

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(Editor: LIU Jia)

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