A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor
- Hengxing Lu1,4,
- Jun Liu1,4,
- Tao Feng2,
- Zihang Guo1,
- Yunjun Yin1,
- Fei Gao1,
- Gengsheng Cao3,
- Xuguang Du1 and
- Sen Wu1
- 1State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China;
- 2Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu 610200, China;
- 3Henan Engineering Laboratory for Mammary Bioreactor, School of Life Science, Henan University, Kaifeng 475004, China
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↵4 These authors contributed equally to this work.
Abstract
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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.271312.120.
- Received September 3, 2020.
- Accepted March 30, 2021.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
