数字农科院2.0

A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor

文献类型： 外文期刊

作者： Lu Hengxing;Liu Jun;Feng Tao;Guo Zihang;Yin Yunjun;Gao Fei;Cao Gengsheng;Du Xuguang;Wu Sen

作者机构：

期刊名称： Genome research

ISSN： 1549-5469

年卷期： 2021 年 31 卷 5 期

页码：

收录情况： JCR（2021版）

摘要： 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. © 2021 Lu et al.; Published by Cold Spring Harbor Laboratory Press.

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