Low-temperature incubation improves both knock-in and knock-down efficiencies by the CRISPR/Cas9 system in Xenopus laevis as revealed by quantitative analysis

• Published in: Biochemical and biophysical research communications 2021-03, Vol.543, p.50-55
• Main Authors: Kato, Sumika, Fukazawa, Taro, Kubo, Takeo
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; Flow cytometry; ICE analysis; Knock-down; Knock-in; Xenopus laevis
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.11.038

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. •We utilized ICE analysis/flow cytometry to quantify gene editing efficiency.•Low-temperature incubation improves the CRISPR/Cas9-mediated knock-down efficiency.•Low-temperature incubation improves the CRISPR/Cas9-mediated knock-in efficiency.