Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation

a.
The Jackson Laboratory, Bar Harbor, ME 04609, USA
b.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

More Information

Corresponding author: E-mail address: wanghaoyi@ioz.ac.cn (Haoyi Wang)

摘要

- /
- /
Abstract: Previously we established Zygote Electroporation of Nucleases (ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models. However, there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol. In this study, we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation. Using this approach, we were able to introduce precise nucleotide substitutions, large segment deletion and short segment insertion into targeted loci with high efficiency.
- CRISPR-Cas9 /
- Electroporation /
- Mouse zygote

HTML全文

参考文献(22)

[1]	Aida, T., Chiyo, K., Usami, T. et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice Genome Biol., 16 (2015),p. 87
[2]	Bassett, A.R., Liu, J.L. J. Genet. Genomics, 41 (2014),pp. 7-19
[3]	Byers, S.L., Payson, S.J., Taft, R.A. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs) Theriogenology, 65 (2006),pp. 1716-1726
[4]	Chapman, K.M., Medrano, G.A., Jaichander, P. et al. Targeted germline modifications in rats using CRISPR/Cas9 and spermatogonial stem cells Cell Rep., 10 (2015),pp. 1828-1835
[5]	Chen, S., Sanjana, N.E., Zheng, K. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis Cell, 160 (2015),pp. 1246-1260
[6]	Cong, L., Ran, F.A., Cox, D. et al. Multiplex genome engineering using CRISPR/Cas systems Science, 339 (2013),pp. 819-823
[7]	Hai, T., Teng, F., Guo, R. et al. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system Cell Res., 24 (2014),pp. 372-375
[8]	Hashimoto, M., Takemoto, T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing Sci. Rep., 5 (2015),p. 11315
[9]	Kaneko, T., Sakuma, T., Yamamoto, T. et al. Simple knockout by electroporation of engineered endonucleases into intact rat embryos Sci. Rep., 4 (2014),p. 6382
[10]	Liang, X., Potter, J., Kumar, S. et al. J. Biotechnol., 208 (2015),pp. 44-53
[11]	Liu, D., Wang, Z., Xiao, A. et al. Efficient gene targeting in zebrafish mediated by a zebrafish-codon-optimized cas9 and evaluation of off-targeting effect J. Genet. Genomics, 41 (2014),pp. 43-46
[12]	Ma, Y., Zhang, X., Shen, B. et al. Generating rats with conditional alleles using CRISPR/Cas9 Cell Res., 24 (2014),pp. 122-125
[13]	Mali, P., Yang, L., Esvelt, K.M. et al. Science, 339 (2013),pp. 823-826
[14]	Niu, Y., Shen, B., Cui, Y. et al. Cell, 156 (2014),pp. 836-843
[15]	Qin, W., Dion, S.L., Kutny, P.M. et al. Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease Genetics, 200 (2015),pp. 423-430
[16]	Ruan, J., Li, H., Xu, K. et al. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs Sci. Rep., 5 (2015),p. 14253
[17]	Schumann, K., Lin, S., Boyer, E. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins Proc. Natl. Acad. Sci. USA, 112 (2015),pp. 10437-10442
[18]	Wang, H., Yang, H., Shivalila, C.S. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering Cell, 153 (2013),pp. 910-918
[19]	Yang, H., Wang, H., Shivalila, C.S. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering Cell, 154 (2013),pp. 1370-1379
[20]	Yang, L., Guell, M., Niu, D. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs) Science, 350 (2015),pp. 1101-1104
[21]	Yin, H., Xue, W., Chen, S. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype Nat. Biotechnol., 32 (2014),pp. 551-553
[22]	Zou, Q., Wang, X., Liu, Y. et al. Generation of gene-target dogs using CRISPR/Cas9 system J. Mol. Cell Biol., 7 (2015),pp. 580-583