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Homology-Mediated End Joining-Based Strategy Shows Robust DNA Targeted Integration Efficiency in Vitro and in Vivo

May 22, 2017     Email"> PrintText Size

A recent study conducted by Dr. YANG Hui’s Lab in collaboration with colleagues at the Institute of Neuroscience of Chinese Academy of Sciences (CAS) demonstrated that homology-mediated end joining (HMEJ)-based strategy can induce robust and efficient DNA targeted integration using CRISPR/Cas9. This study was published in Cell Research.

This work successfully devised an HMEJ-based strategy that showed higher knock-in efficiencies than existing strategies in multiple systems, including cultured cells, embryos and tissues in vivo. With higher editing efficiency and better fidelity compared to the NHEJ-based method, the HMEJ-based method holds a great promise for applications such as generation of gene-modified animal models and in vivo targeted gene replacement therapy.

Precisely targeted genome editing is highly desired for clinical applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system greatly facilitates targeted integration of transgenes by generating a targeted DNA double-strand break (DSB) in the genome. Once a DSB is created, targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ).

The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues. However, NHEJ-based targeted integration introduced random directions in integration and various types of indels at the junctions, making it difficult to construct endogenous and exogenous in-frame fusion genes for chimeric protein production. MMEJ-based targeted integration could only elevate the efficiency in some systems. Thus, researchers examined the possibility that CRISPR/Cas9-mediated DNA cleavage on an HR donor could improve the efficiency of homology-mediated gene integration, especially in non-dividing cells.

In this study, researchers devised a new HMEJ-based strategy using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ~800 bp of homology arms and the targeted genome to improve the efficiency of homology-mediated gene integration especially in non-dividing cells. To test this idea, they compared the knock-in efficiency in many systems using four types of donors: an HMEJ donor (sgRNA target sites plus long HAs (800 bp)), an HR donor (only long HAs), an NHEJ donor (only sgRNA target sites) and an MMEJ donor (sgRNA target sites plus short HAs (20 bp)).

According to the results, no significant improvement of the targeting efficiency was found in either mouse embryonic stem cells or the neuroblastoma cell line N2a, as compared to the HR-based method. However, the HMEJ-based method yielded a higher knockin efficiency in HEK293T cells, primary astrocytes and neurons.

This approach achieved transgene integration in mouse and monkey embryos as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. In addition, researchers explored the mechanism of the HMEJ-based method, and found that it may depend on the HMEJ and HR pathways. The HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

This work was supported by CAS Strategic Priority Research Program, the National Hightech R&D Program, the National Natural Science Foundation of China, China Youth Thousand Talents Program, Breakthrough Project of Chinese Academy of Sciences, the National Key Technology R&D Program of China, Shanghai City Committee of Science and Technology, and CAS Hundreds of Talents Program of China.

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(Editor: LIU Jia)

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