Heritable multiplex genetic engineering in rats using CRISPR/Cas9

Yuanwu Ma; Bin Shen; Xu Zhang; Yingdong Lu; Wei Chen; Jing Ma; Xingxu Huang; Lianfeng Zhang

doi:10.1371/journal.pone.0089413

Heritable multiplex genetic engineering in rats using CRISPR/Cas9

PLoS One. 2014 Mar 5;9(3):e89413. doi: 10.1371/journal.pone.0089413. eCollection 2014.

Authors

Yuanwu Ma¹, Bin Shen², Xu Zhang¹, Yingdong Lu¹, Wei Chen¹, Jing Ma¹, Xingxu Huang², Lianfeng Zhang¹

Affiliations

¹ Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China.
² MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing Biomedical Research Institute, National Resource Center for Mutant Mice, Nanjing, China.

Abstract

The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (ApoE, B2m, Prf1, and Prkdc) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the off-target mutagenesis and mosaicism are rarely detected by comprehensive analysis. Thus, the CRISPR/Cas9 system makes it possible to efficiently and reliably generate gene knock-out rats.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Base Sequence
CRISPR-Associated Proteins / genetics
Clustered Regularly Interspaced Short Palindromic Repeats*
DNA Cleavage
Female
Genetic Engineering*
Male
Microinjections
Molecular Sequence Data
Mosaicism
Plasmids / genetics
Rats, Sprague-Dawley
Rats, Transgenic

Substances

CRISPR-Associated Proteins

Grants and funding

The present work was supported in part by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BA139B02, 2009CB918700) and the Youth Foundation of CAMS & PUMC (2012J25). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.