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.

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

Grant support

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.