Tamoxifen- and Mifepristone-Inducible Versions of CRISPR Effectors, Cas9 and Cpf1

ACS Synth Biol. 2018 Sep 21;7(9):2160-2169. doi: 10.1021/acssynbio.8b00145. Epub 2018 Sep 4.

Abstract

Methods for making specific modifications to the genomes of living cells are powerful research tools. Two methods currently dominate, CRISPR and Cre recombinase. CRISPR has the advantage that it can act on unmodified target genes; Cre has the advantage of being available in drug-inducible versions, allowing temporal control, but it requires engineering ("floxing") of the target gene. Here, we have combined these advantages by constructing drug (tamoxifen/mifepristone)-inducible Cas9 and Cpf1 CRISPR effectors. We demonstrate their low background activity and robust activation with drugs, by using gRNAs to target them to TetR, in a cell carrying a Tet-repressed reporter gene. As well as being useful in their own right, the research tools generated here will pave the way to making further drug-inducible effector proteins.

Keywords: 3D structure; CRISPR; drug inducible; mammalian gene expression systems; molecular engineering; synthetic biology; tetracycline repressor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics*
  • CRISPR-Associated Protein 9 / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Endonucleases / genetics*
  • Gene Editing / methods*
  • Gene Expression / drug effects*
  • Genes, Reporter
  • HEK293 Cells
  • Humans
  • Mifepristone / pharmacology*
  • Mutagenesis
  • Plasmids / genetics
  • Plasmids / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Tamoxifen / pharmacology*

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Receptors, Estrogen
  • Recombinant Fusion Proteins
  • Tamoxifen
  • Mifepristone
  • CRISPR-Associated Protein 9
  • Cas12a protein
  • Endonucleases