CasX enzymes comprise a distinct family of RNA-guided genome editors

Nature. 2019 Feb;566(7743):218-223. doi: 10.1038/s41586-019-0908-x. Epub 2019 Feb 4.

Abstract

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / classification*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Associated Proteins / ultrastructure*
  • CRISPR-Cas Systems / genetics*
  • Cryoelectron Microscopy
  • DNA / chemistry
  • DNA / metabolism
  • DNA / ultrastructure
  • DNA Cleavage
  • Escherichia coli / genetics
  • Evolution, Molecular
  • Gene Editing*
  • Gene Silencing
  • Genome, Bacterial / genetics
  • Genome, Human / genetics
  • Humans
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Domains
  • RNA, Guide / metabolism

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • DNA