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

  • Author(s): Liu, Jun-Jie
  • Orlova, Natalia
  • Oakes, Benjamin L
  • Ma, Enbo
  • Spinner, Hannah B
  • Baney, Katherine LM
  • Chuck, Jonathan
  • Tan, Dan
  • Knott, Gavin J
  • Harrington, Lucas B
  • Al-Shayeb, Basem
  • Wagner, Alexander
  • Brötzmann, Julian
  • Staahl, Brett T
  • Taylor, Kian L
  • Desmarais, John
  • Nogales, Eva
  • Doudna, Jennifer A
  • et al.
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.

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