Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan.
Biochem Biophys Res Commun. 2021 Mar 5;543:50-55. doi: 10.1016/j.bbrc.2020.11.038. Epub 2021 Jan 27.
The recent development of the CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. Gene-edited F0 individuals created by these methods, however, are mosaics with both mutated/knocked-in and unedited wild-type cells, and therefore precise determination and higher efficiency of knock-down and knock-in methods are desirable, especially for analyses of F0 individuals. To clarify the ratio of cells that are gene-edited by CRISPR/Cas9 methods to the whole cells in F0 individuals, we subjected Inference of CRISPR Edits analysis for knock-down experiments and flow cytometry for knock-in experiments to the F0 individuals. With these quantitative methods, we showed that low-temperature incubation of X. laevis embryos after microinjection improved the mutation rate in the individuals. Moreover, we applied low-temperature incubation when using a knock-in method with long single-strand DNA and found improved knock-in efficiency. Our results provide a simple and useful way to evaluate and improve the efficiency of gene editing in X. laevis.
CRISPR/Cas9 介导的基因编辑技术的最新发展为非洲爪蟾（Xenopus laevis）提供了各种基因敲低和敲入方法。然而，这些方法创造的 F0 个体是突变/敲入和未编辑野生型细胞的嵌合体，因此需要更精确和高效的敲低和敲入方法，特别是对于 F0 个体的分析。为了明确 CRISPR/Cas9 方法编辑的基因在 F0 个体中的细胞比例，我们对 F0 个体进行了敲低实验的 Inference of CRISPR Edits 分析和敲入实验的流式细胞术分析。通过这些定量方法，我们表明，在微注射后低温孵育 X. laevis 胚胎可提高个体中的突变率。此外，当使用带有长单链 DNA 的敲入方法时，我们应用了低温孵育，发现敲入效率得到了提高。我们的结果提供了一种简单而有用的方法来评估和提高 X. laevis 中基因编辑的效率。
