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

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

Author information

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.

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PMID:
30718774
DOI:
10.1038/s41586-019-0908-x

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