One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering
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Jaenisch_One-step.pdf
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Author(s)
Yang, Hui
Wang, Haoyi
Cheng, Albert W.
Shi, Linyu
Shivalila, Chikdu Shakti
Jaenisch, Rudolf
Date Issued
August 2013
Journal
Cell
Publisher
Elsevier
Citation
Yang, Hui; Wang, Haoyi; Shivalila, Chikdu S.; Cheng, Albert W.; Shi, Linyu and Jaenisch, Rudolf. "One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering." Cell 154, no.6 (September 2013): 1370–1379 © 2013 Elsevier Inc
Version
Author's final manuscript
Abstract
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
MIT Department
Massachusetts Institute of Technology. Computational and Systems Biology Program
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/j.cell.2013.08.022
