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Cited by 446CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

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

Open access
Liu, Jun-Jie, Orlova, Natalia, Oakes, Benjamin L., Ma, Enbo, Spinner, Hannah B., Baney, Katherine L. M., Chuck, Jonathan, Tan, Dan +10 more·2019·Nature
DOI 10.1038/s41586-019-0908-xPMC PMC6662743
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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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  • Smaller genome editing proteins, such as S. aureus Cas9, C. jejuni Cas9 and newly identified CRISPR-Cas enzymes, may circumvent the AAV cargo limit.

    “Smaller genome editing proteins, such as the S. aureus Cas9, C. jejuni Cas9 and newly identified CRISPR-Cas enzymes, may circumvent this issue 23,61-65 .”

    From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [23]

    The Conclusions (page unavailable) say CasX’s compact size may offer advantages for therapeutic delivery, supporting the claim’s point about a newly identified, smaller genome-editing enzyme. Our copy does not specifically discuss the AAV cargo limit or establish the claim for S. aureus Cas9 and C. jejuni Cas9. (LLM, conf=0.84)

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Authors
Liu, J.-J., Orlova, N., Oakes, B. L., Ma, E., Spinner, H. B., Baney, K. L. M., Chuck, J., Tan, D., Knott, G. J., Harrington, L. B., Al-Shayeb, B., Wagner, A., Brötzmann, J., Staahl, B. T., Taylor, K. L., Desmarais, J., Nogales, E., & Doudna, J. A.
Affiliations
  • QB3 (US)
  • Innovative Genomics Institute (US)
  • University of California, Berkeley (US)
  • Salk Institute for Biological Studies (US)
  • Max Planck Institute of Biochemistry (DE)
  • Ludwig-Maximilians-Universität München (DE)
  • Gladstone Institutes (US)
Corresponding author
  • Nogales, Eva
  • Doudna, Jennifer A.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2019-02
Volume
566
Issue
7743
Pages
218-223
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-019-0908-x
PMC
PMC6662743
OpenAlex
W2911354827
Cited by
446
References
6
Citations by year
2026: 29 · 2025: 31 · 2024: 47 · 2023: 57 · 2022: 76 · 2021: 71 · 2020: 77 · 2019: 56
FWCI
23.15
Type
journal article
Language
en
Update notices
correction (2019-04-03)
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • National Science Foundation (1244557, P01GM051487)
  • Howard Hughes Medical Institute
  • National Institutes of Health
  • National Human Genome Research Institute (RM1HG009490)
  • National Institute of General Medical Sciences (P50GM082250, P01GM051487)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
CRISPR-Cas, RNA-guided genome editing, Cas9, CRISPR/Cas12a, adaptive immunity, double-stranded DNA cleavage, guide RNA, DNA unwinding, convergent evolution, Escherichia coli, human genome modification, cryo-EM structure
MeSH terms
Protein Domains, Gene Editing, RNA, Guide, CRISPR-Cas Systems, DNA, Escherichia coli, Humans, Models, Molecular, Nucleic Acid Conformation, Genome, Human, Genome, Bacterial, RNA, Guide, Kinetoplastida, Evolution, Molecular, Cryoelectron Microscopy, Gene Silencing, DNA Cleavage, CRISPR-Cas Systems, CRISPR-Associated Proteins

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