Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

PLoS One. 2016 Nov 10;11(11):e0166020. doi: 10.1371/journal.pone.0166020. eCollection 2016.

Abstract

CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Mutagenesis*
  • Promoter Regions, Genetic
  • RNA, Catalytic / metabolism*
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Transgenes
  • Zebrafish / embryology
  • Zebrafish / genetics*

Substances

  • RNA, Catalytic
  • RNA, Guide, CRISPR-Cas Systems

Grants and funding

This work was supported by the Agency for Science Technology and Research (A*STAR) and by the Toh Kian Chui Foundation to PWI. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.