Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.
The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China.
Mol Biol Rep. 2021 Nov;48(11):7325-7332. doi: 10.1007/s11033-021-06734-x. Epub 2021 Oct 26.
Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present research aimed to establish a rapid, sensitive and visual method for genotyping of germline genome-edited mutants with small genomic fragment deletion.
The genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limit of detection (LOD) of 0.1 ng.
The proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-edited mutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.
基因组编辑技术结合人工核酸酶快速发展，使众多生物的基因组 DNA 得到高效、精确的修饰。本研究旨在建立一种快速、灵敏、可视化的方法，用于对具有小基因组片段缺失的生殖系基因组编辑突变体进行基因分型。
使用 TALEN 系统生成的肌生成抑制素（MSTN）基因 2 个碱基缺失和 11 个碱基缺失的基因组编辑猪作为试验材料，验证了所提出的等位基因特异性 PCR（AS-PCR）和侧流核酸生物传感器（LFNAB）级联方法。AS-PCR 可以产生带有不同标签的产物，以区分基因组编辑等位基因和野生型等位基因。应用 LFNAB 对 AS-PCR 产物进行可视化检测，无需使用额外的仪器。此外，我们证明 AS-PCR 和 LFNAB 级联可以准确、直观地区分肌生成抑制素（MSTN）基因小基因组片段缺失的基因组编辑猪和野生型猪，检测限（LOD）为 0.1ng。
所提出的 AS-PCR 和 LFNAB 级联可用于快速可视化地对具有小基因组片段缺失的基因组编辑突变体进行基因分型，为基因组编辑动物基因分型提供了一个平台。
