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

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. (c) 2020 Elsevier Inc. All rights reserved.

Automated summary

Recent development of CRISPR/Cas9-mediated gene editing technique has provided various gene knock-down and knock-in methods for Xenopus laevis. However, F0 individuals created by these methods are mosaics, necessitating precise determination and higher efficiency. Low-temperature incubation improved mutation rate and knock-in efficiency. These results offer a simple and useful way to evaluate and enhance gene editing efficiency in Xenopus laevis.