State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu 610200, China.
Genome Res. 2021 May;31(5):900-909. doi: 10.1101/gr.271312.120. Epub 2021 Apr 1.
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.
在模式生物中进行靶向诱变对于基因功能注释和生物医学研究至关重要。尽管 CRISPR-Cas9 系统在基因编辑方面取得了技术进步，但在大型动物模型中快速有效地引入定点突变仍然是一个挑战。在这里，我们开发了一种强大且灵活的插入诱变策略，即非同源性靶向捕获（HIT 捕获），该策略通用，能够高效地靶向捕获感兴趣的内源性基因，而无需同源臂和胚胎干细胞。进一步优化并为 HIT 捕获供体配备了特定的 DNA 反转机制，使在单个实验中一步生成可逆和条件性等位基因成为可能。作为概念验证，我们成功地在原代猪成纤维细胞中为 21 个与疾病相关的基因创建了突变等位基因，平均敲入频率为 53.2%，比以前的方法有了很大的提高。这里提出的多功能 HIT 捕获策略有望简化突变等位基因的靶向生成，并促进猪等大型哺乳动物的大规模诱变。
