Synthetically Modified Guide RNA and Donor DNA Are a Versatile Platform for CRISPR-Cas9 Engineering

Elife. 2017 May 2;6:e25312. doi: 10.7554/eLife.25312.

Abstract

Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5' fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.

Keywords: CRISPR/Cas9; chromosomes; drug delivery; gene editing; genes; human biology; medicine; none.

MeSH terms

  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • DNA / chemistry*
  • DNA / genetics*
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • RNA, Guide / chemistry*
  • RNA, Guide / genetics*

Substances

  • Bacterial Proteins
  • RNA, Guide
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Cpf1 nuclease, Acidaminococcus
  • Endonucleases

Grant support

The authors declare that there was no funding for this work