CasX enzymes comprise a distinct family of RNA-guided genome editors
Abstract
The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against bacteriophage and function as powerful tools for genome editing in wide-ranging cell types. Here we present a third and fundamentally distinct RNA-guided platform, CRISPR-CasX, which uses a unique structure and mechanism for programmable double-stranded DNA cleavage. Biochemical and in vivo data demonstrate that CasX is active for E. coli and human genome modification. Eight cryo-EM structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and an unanticipated 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.
- Authors:
-
- Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences
- Univ. of California, Berkeley, CA (United States). Innovative Genomics Inst.
- Univ. of California, Berkeley, CA (United States)
- Salk Institute for Biological Studies, La Jolla, CA (United States). Clayton Foundation Laboratories of Peptide Biology
- Max-Planck Inst. for Biochemistry, Planegg (Germany)
- Ludwig-Maximilians-Univ., Munich (Germany)
- Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences, and Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences, Howard Hughes Medical Inst., and Innovative Genomics Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC); National Science Foundation (NSF); National Institutes of Health (NIH)
- OSTI Identifier:
- 1605229
- Grant/Contract Number:
- AC02-05CH11231; P01GM051487; P50GM082250; 1244557
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature (London)
- Additional Journal Information:
- Journal Name: Nature (London); Journal Volume: 566; Journal Issue: 7743; Journal ID: ISSN 0028-0836
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Liu, Jun-Jie, Orlova, Natalia, Oakes, Benjamin L., Ma, Enbo, Spinner, Hannah B., Baney, Katherine L. M., 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, and Doudna, Jennifer A. CasX enzymes comprise a distinct family of RNA-guided genome editors. United States: N. p., 2019.
Web. doi:10.1038/s41586-019-0908-x.
Liu, Jun-Jie, Orlova, Natalia, Oakes, Benjamin L., Ma, Enbo, Spinner, Hannah B., Baney, Katherine L. M., 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. CasX enzymes comprise a distinct family of RNA-guided genome editors. United States. https://doi.org/10.1038/s41586-019-0908-x
Liu, Jun-Jie, Orlova, Natalia, Oakes, Benjamin L., Ma, Enbo, Spinner, Hannah B., Baney, Katherine L. M., 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, and Doudna, Jennifer A. Mon .
"CasX enzymes comprise a distinct family of RNA-guided genome editors". United States. https://doi.org/10.1038/s41586-019-0908-x. https://www.osti.gov/servlets/purl/1605229.
@article{osti_1605229,
title = {CasX enzymes comprise a distinct family of RNA-guided genome editors},
author = {Liu, Jun-Jie and Orlova, Natalia and Oakes, Benjamin L. and Ma, Enbo and Spinner, Hannah B. and Baney, Katherine L. M. and Chuck, Jonathan and Tan, Dan and Knott, Gavin J. and Harrington, Lucas B. and Al-Shayeb, Basem and Wagner, Alexander and Brötzmann, Julian and Staahl, Brett T. and Taylor, Kian L. and Desmarais, John and Nogales, Eva and Doudna, Jennifer A.},
abstractNote = {The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against bacteriophage and function as powerful tools for genome editing in wide-ranging cell types. Here we present a third and fundamentally distinct RNA-guided platform, CRISPR-CasX, which uses a unique structure and mechanism for programmable double-stranded DNA cleavage. Biochemical and in vivo data demonstrate that CasX is active for E. coli and human genome modification. Eight cryo-EM structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and an unanticipated 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.},
doi = {10.1038/s41586-019-0908-x},
journal = {Nature (London)},
number = 7743,
volume = 566,
place = {United States},
year = {Mon Feb 04 00:00:00 EST 2019},
month = {Mon Feb 04 00:00:00 EST 2019}
}
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Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency
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Increasing the specificity of CRISPR systems with engineered RNA secondary structures
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Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era
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Multiplex genome editing of microorganisms using CRISPR-Cas
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