Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis.
Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Zhaoying
Z
Shi
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Fengqin
F
Wang
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Yan
Y
Cui
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Zhongzhen
Z
Liu
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Xiaogang
X
Guo
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Yanqi
Y
Zhang
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China.
Yi
Y
Deng
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China chenyl@sustc.edu.cn zhaohui@cuhk.edu.hk.
Hui
H
Zhao
*Chinese Academy of Sciences Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China; Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China; School of Life Sciences, Anhui University, Hefei, Anhui, China; University of Chinese Academy of Sciences, Beijing, China; School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, Guangdong, China chenyl@sustc.edu.cn zhaohui@cuhk.edu.hk.
Yonglong
Y
Chen
D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D014981:Xenopus / Q000196:embryology / Q000235:genetics*
concurrent cleavage; homology independent; knock-in