Wednesday, November 27, 2013

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA - Ikawa lab papers, November 2013.

The first of these 2 papers is the first report of mouse mutagenesis by pronuclear injection of circular plasmids expressing CRISPR gRNA and Cas9 mRNA.    In a follow-up paper, the authors demonstrate this approach on many more genes; their approach is to validate the CRISPR plasmids in cell transfections first, then use the effective ones for zygote injections.

The plasmid used in both papers was PX330, which was made by the Zhang lab - links for this are at the bottom.   

1.  Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.

Daisuke Mashiko, Yoshitaka Fujihara, Yuhkoh Satouh, Haruhiko Miyata, Ayako Isotani & Masahito Ikawa

Scientific Reports
Article number:
3 Accepted 12 November 2013 Published
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.
 2014 Jan;56(1):122-9. doi: 10.1111/dgd.12113. Epub 2013 Dec 26.

Links for PX330 and PX335:  

PX335 is similar to PX330 except PX335 has a nickase form of Cas9.

Friday, November 1, 2013

Key first papers demonstrating CRISPR in mouse embryos

Wang, H., H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, and R. Jaenisch, One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell, 2013. 153(4): p. 910-8.    First paper showing mutagenesis. including multiplexing, in mouse embryos by pronuclear injection. Remarkably high efficiencies reported.

Li, D., Z. Qiu, Y. Shao, Y. Chen, Y. Guan, M. Liu, Y. Li, N. Gao, L. Wang, X. Lu, and Y. Zhao, Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(8): p. 681-3.

Fujii, W., K. Kawasaki, K. Sugiura, and K. Naito, Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res, 2013. 41(20): p. e187.    By "large-scale", they mean a deletion of several kilobases.

Shen, B., J. Zhang, H. Wu, J. Wang, K. Ma, Z. Li, X. Zhang, P. Zhang, and X. Huang, Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research, 2013. 23(5): p. 720-3.

Yang, H., H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, and R. Jaenisch, One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013. 154(6): p. 1370-9.   First paper showing highly efficient CRISPR HDR-mediated gene editing in mouse embryos, in contrast to simple mutagenesis via the NHEJ pathway.