ANDROFEST 2014* CRISPR/Cas mediated genome editing in mice and its application for reproductive studies (#58)
Targeted gene manipulation through homologous recombination in embryonic stem (ES) cells and subsequent chimeric mouse production is a powerful method and conventionally used worldwide. However it is laborious, costly, and time consuming. Moreover, only well trained researchers are able to accomplish the experimental procedures. The emergence of recombinant artificial endonucleases, ZFN and TALENS, has opened the window for the next generation targeted mutagenesis. The recombinant artificial endonuclease causes double strand break (DSB) then subsequent error-prone non-homologous end joying (NHEJ) would result in small indels. More recently, the recombinant CRISPR/CAS system was developed. The combination of Cas9 protein with guide RNA (gRNA) could reconstitute RNA-based nucleases, and thus prepared nucleoprotein functions as a designed endonuclease. The targeted gene editing in mammalian zygote has also been demonstrated. Here we present that the pronuclear injection of the circular plasmid expressing hCas9 and sgRNA efficiently generates gene manipulated mice. Among the 196 pups developed from 2,397 eggs injected, 100 were found to be targeted gene manipulated animals. Moreover, except 4 mice carrying Y chromosome mutation, 47 out of 96 carried biallelic mutations 1, 2. The application of our approach for the study of reproduction will also be discussed.
- Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M. Sci Rep. 2013 Nov 27;3:3355. doi: 10.1038/srep03355.
- Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Mashiko D, Young SA, Muto M, Kato H, Nozawa K, Ogawa M, Noda T, Kim YJ, Satouh Y, Fujihara Y, Ikawa M. Dev Growth Differ. 2014 Jan;56(1):122-9. doi: 10.1111/dgd.12113.
