Genome Editing Research Center, KRIBB, Daejeon (34141), Republic of Korea; Department of Bio-molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon (34113), Republic of Korea.
Genome Editing Research Center, KRIBB, Daejeon (34141), Republic of Korea; Department of Bio-molecular Science, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon (34113), Republic of Korea; GenKOre, Daejeon (34141), Republic of Korea.
Mol Cell Probes. 2021 Feb;55:101675. doi: 10.1016/j.mcp.2020.101675. Epub 2020 Nov 25.
Base editors and prime editors induce precise DNA modifications over one or several nucleotides in eukaryotic cells. The T7E1 assay has been widely adopted for the assessment of genome editing, but it has several limitations in the applications for prime editing and base editing due to low sensitivity, inaccuracy and additional disadvantages. Here, we propose a short inner primer-assisted, tetra primer-paired amplification (SIPATA) method as an alternative to T7E1 analysis. SIPATA is a PCR-based method in which two long outer and two short (15 nt) inner primers are used for the amplification of a specific genotype in the presence of Hot start-Taq. One of the inner primers carries a 3'-terminally wild-type nucleotide sequence, and the other carries a post-editing sequence. Under optimized conditions, SIPATA enabled sensitive and accurate genotyping of single-nucleotide conversions by base editors and prime editors. Furthermore, SIPATA could be applied to trace low levels of DNA modifications achieved by HDR-mediated gene correction or chimerism during the generation of model animals. Multiplexed genotyping was also possible without compromising those multifaceted analytical advantages of SIPATA. Our findings demonstrate that SIPATA offers a robust, fast and sensitive genotyping platform for single-nucleotide variations in a variety of CRISPR applications.
碱基编辑器和先导编辑酶可在真核细胞中精确地编辑一个或几个核苷酸。T7E1 分析已被广泛用于基因组编辑的评估，但由于灵敏度低、准确性差和其他缺点，在先导编辑和碱基编辑的应用中存在一些局限性。在这里，我们提出了一种短内引物辅助、四引物配对扩增（SIPATA）方法作为 T7E1 分析的替代方法。SIPATA 是一种基于 PCR 的方法，其中在外引物较长的情况下，使用两个长外引物和两个短（15nt）内引物在 Hot start-Taq 的存在下扩增特定基因型。其中一个内引物携带 3'-末端野生型核苷酸序列，另一个携带编辑后序列。在优化条件下，SIPATA 能够敏感和准确地对碱基编辑器和先导编辑酶引起的单核苷酸转换进行基因分型。此外，SIPATA 可用于追踪模型动物生成过程中 HDR 介导的基因校正或嵌合体中低水平的 DNA 修饰。多plex 基因分型也可以在不影响 SIPATA 多方面分析优势的情况下进行。我们的研究结果表明，SIPATA 为各种 CRISPR 应用中的单核苷酸变异提供了一种强大、快速和敏感的基因分型平台。
