A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Cell. 2021 Feb 4;184(3):675-688.e19. doi: 10.1016/j.cell.2020.12.017. Epub 2021 Jan 8.


CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

Keywords: CRISPR-Cas; Cas9; autoimmunity; bacterial regulation; bacteriophage; sgRNA; tracr-L; tracrRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoimmunity / genetics
  • Base Sequence
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Conserved Sequence
  • Down-Regulation / genetics
  • Gene Expression*
  • Homeostasis / genetics*
  • Models, Genetic
  • Mutation / genetics
  • Operon / genetics
  • Promoter Regions, Genetic / genetics
  • RNA, Guide, Kinetoplastida / genetics*
  • Streptococcus pyogenes / genetics
  • Stress, Physiological / genetics
  • Transcription, Genetic
  • Transcriptional Activation / genetics


  • RNA, Guide
  • CRISPR-Associated Protein 9