One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

• Published in: Cell research Vol. 27; no. 7; pp. 933 - 945
• Main Authors: Zuo, Erwei, Cai, Yi-Jun, Li, Kui, Wei, Yu, Wang, Bang-An, Sun, Yidi, Liu, Zhen, Liu, Jiwei, Hu, Xinde, Wei, Wei, Huo, Xiaona, Shi, Linyu, Tang, Cheng, Liang, Dan, Wang, Yan, Nie, Yan-Hong, Zhang, Chen-Chen, Yao, Xuan, Wang, Xing, Zhou, Changyang, Ying, Wenqin, Wang, Qifang, Chen, Ren-Chao, Shen, Qi, Xu, Guo-Liang, Li, Jinsong, Sun, Qiang, Xiong, Zhi-Qi, Yang, Hui
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-07-2017; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/1513/1967/3196; 631/1647/767/722; 631/45/500; Animal models; Animals; ARNTL Transcription Factors - genetics; Bacterial Proteins; Biomedical and Life Sciences; Cell Biology; Chromosome deletion; Clonal deletion; CRISPR; CRISPR-Associated Protein 9; CRISPR-Cas Systems - genetics; Embryo, Mammalian - cytology; Embryos; Endonucleases; Exons - genetics; Gene deletion; Gene Editing - methods; Gene Knockout Techniques; Genes; Genetic modification; Genome editing; Haplorhini - genetics; Life Sciences; Membrane Proteins - genetics; Mice; Mice, Inbred C57BL; Mice, Knockout; Monkeys; Mosaicism; Mosaicism - embryology; mRNA; Oocyte Retrieval; Original; original-article; Phenotype; RNA, Guide, Kinetoplastida - genetics; RNA, Messenger - genetics; Rodents; Whole Genome Sequencing; Y Chromosome; Y chromosomes; Zygote - cytology; CRISPR/Cas9; multiple sgRNAs; complete gene knockout
• ISSN: 1001-0602; 1748-7838
• DOI: 10.1038/cr.2017.81

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs （spaced 10-200 bp apart） that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficien- cies （100% on Arnt[ and 91% on Prrt2） in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.