Generation of site-specific mutant mice using the CRISPR/Cas9 system.
The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.
1. Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China; 2. Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China;
Min
M
Bai
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
Qi
Q
Li
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
Yan-jiao
YJ
Shao
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
Yuan-hua
YH
Huang
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
Da-li
DL
Li
Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
Yan-lin
YL
Ma
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods*; D056484:Jumonji Domain-Containing Histone Demethylases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D059767:Recombinational DNA Repair; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D044783:F-Box Proteins; D010105:Oxygenases; D056484:Jumonji Domain-Containing Histone Demethylases; C524747:Kdm2b protein, mouse; C020066:dimethylaniline monooxygenase (N-oxide forming)