CRISPR/Cas9 mediated genome editing in mice and its application for the study of reproduction
CRISPR/Cas9 system has opened the new era for reverse genetics. In 2013, we developed the efficient gene knockout system in mice by injecting the plasmids expressing humanized Cas9 (hCas9) and single guide RNA (sgRNA) into zygotes. Now it is replaced by electroporation of oocytes with CAS9/crRNA/tracrRNA ribonucleoprotein complex. To date, we have knocked out 170 testis abundant genes and analyzed the phenotypes in vivo. Whereas 105 of the KO mouse lines were fertile and did not show any drastic phenotypes, 2 KO mouse lines showed lethality. The remaining 63 KO mouse lines showed infertility or subfertility and propelled our in vivo study of reproduction.
However, the mosaicism in founder generation complicated the genotyping and phenotyping analysis. Therefore, we applied CRISPR/Cas9 mediated genome editing in ES cells. In addition to the indels, the complicated genome editing such as large deletion, point mutation, and knockin was efficient in ES cells. The combination of ES mediated genome editing and chimeric analysis in founder generation would provide an alternative strategy for gene function study in vivo.
Our approaches are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.
Dr. Masahito Ikawa is Distinguished Professor of the Research Institute for Microbial Diseases and Director of the Animal Resource Center for Infectious Diseases at Osaka University. His doctoral dissertation at Osaka University involved the application of green fluorescent protein (GFP) as a marker in transgenic mice, and he was the first to generate “green mice” expressing GFP ubiquitously. He performed postdoctoral studies at the Salk Institute in San Diego and used lentiviral vectors for transgenesis and gene therapy studies. As a Research Associate, Associate Professor, and Professor at Osaka University, he has been studying male and female fertility through gene-manipulated mice. In these capacities, Dr. Ikawa runs an active transgenic technology program in his laboratory, Institute, and Center that includes the use of CRISPR/Cas9 to produce knockout and knockin mice to study male and female infertility.