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

Jun-Jie Liu; Natalia Orlova; Benjamin L Oakes; Enbo Ma; Hannah B Spinner; Katherine L M Baney; Jonathan Chuck; Dan Tan; Gavin J Knott; Lucas B Harrington; Basem Al-Shayeb; Alexander Wagner; Julian Brötzmann; Brett T Staahl; Kian L Taylor; John Desmarais; Eva Nogales; Jennifer A Doudna

doi:10.1038/s41586-019-0908-x

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.

Authors

Jun-Jie Liu^{1

2

3}, Natalia Orlova², Benjamin L Oakes⁴, Enbo Ma¹, Hannah B Spinner⁴, Katherine L M Baney⁴, Jonathan Chuck¹, Dan Tan⁵, Gavin J Knott¹, Lucas B Harrington¹, Basem Al-Shayeb⁶, Alexander Wagner⁷, Julian Brötzmann⁸, Brett T Staahl^{1

4}, Kian L Taylor⁴, John Desmarais⁴, Eva Nogales^{9

10

11

12}, Jennifer A Doudna^{13

14

15

16

17

18}

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
² California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA.
³ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, CA, USA.
⁵ Clayton Foundation Laboratories of Peptide Biology, Salk Institute for Biological Studies, La Jolla, CA, USA.
⁶ Department of Plant and Microbiology, University of California, Berkeley, CA, USA.
⁷ Max-Planck-Institute for Biochemistry, Planegg, Germany.
⁸ Faculty of Chemistry and Pharmacy, Ludwig-Maximilians-University, Munich, Germany.
⁹ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. enogales@lbl.gov.
¹⁰ California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA. enogales@lbl.gov.
¹¹ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. enogales@lbl.gov.
¹² Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. enogales@lbl.gov.
¹³ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁴ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁵ Innovative Genomics Institute, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁶ Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁷ Department of Chemistry, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁸ Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.

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, CRISPR-Cas Systems / metabolism

Substances

CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding