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

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.

doi:10.1038/s41586-019-0908-x

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

Journal Article · 03 February 2019 · Nature (London)

DOI: https://doi.org/10.1038/s41586-019-0908-x · OSTI ID:1605229

Liu, Jun-Jie ^[1]; Orlova, Natalia ^[2]; Oakes, Benjamin L. ^[3]; Ma, Enbo ^[4]; Spinner, Hannah B. ^[3]; Baney, Katherine L. M. ^[3]; Chuck, Jonathan ^[4]; Tan, Dan ^[5]; Knott, Gavin J. ^[4]; Harrington, Lucas B. ^[4]; Al-Shayeb, Basem ^[4]; Wagner, Alexander ^[6]; Brötzmann, Julian ^[7]; Staahl, Brett T. ^[3]; Taylor, Kian L. ^[3]; Desmarais, John ^[3]; Nogales, Eva ^[8]; Doudna, Jennifer A. ^[9]

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)

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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1605229

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7743 Vol. 566; ISSN 0028-0836

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Visualisation of dCas9 target search in vivo using an open-microscopy framework Martens, Koen J. A.; van Beljouw, Sam P. B.; van der Els, Simon Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-11514-0	journal	August 2019
The emerging and uncultivated potential of CRISPR technology in plant science Zhang, Yingxiao; Malzahn, Aimee A.; Sretenovic, Simon Nature Plants, Vol. 5, Issue 8 https://doi.org/10.1038/s41477-019-0461-5	journal	July 2019
Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing Cheng, Qiang; Wei, Tuo; Farbiak, Lukas Nature Nanotechnology, Vol. 15, Issue 4 https://doi.org/10.1038/s41565-020-0669-6	journal	April 2020
Increasing the specificity of CRISPR systems with engineered RNA secondary structures Kocak, D. Dewran; Josephs, Eric A.; Bhandarkar, Vidit Nature Biotechnology, Vol. 37, Issue 6 https://doi.org/10.1038/s41587-019-0095-1	journal	April 2019
Leveraging implicit knowledge in neural networks for functional dissection and engineering of proteins Upmeier zu Belzen, Julius; Bürgel, Thore; Holderbach, Stefan Nature Machine Intelligence, Vol. 1, Issue 5 https://doi.org/10.1038/s42256-019-0049-9	journal	May 2019
Direct observation of the formation of a CRISPR–Cas12a R-loop complex at the single-molecule level Cui, Yang; Tang, Yangchao; Liang, Meng Chemical Communications, Vol. 56, Issue 14 https://doi.org/10.1039/c9cc08325a	journal	January 2020
Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a Swartjes, Thomas; Staals, Raymond H. J.; van der Oost, John Biochemical Society Transactions, Vol. 48, Issue 1 https://doi.org/10.1042/bst20190563	journal	December 2019
Making the cut(s): how Cas12a cleaves target and non-target DNA Swarts, Daan C. Biochemical Society Transactions, Vol. 47, Issue 5 https://doi.org/10.1042/bst20190564	journal	October 2019
Multiplex genome editing of microorganisms using CRISPR-Cas Adiego-Pérez, Belén; Randazzo, Paola; Daran, Jean Marc FEMS Microbiology Letters, Vol. 366, Issue 8 https://doi.org/10.1093/femsle/fnz086	journal	April 2019
PAM recognition by miniature CRISPR nucleases triggers programmable double-stranded DNA target cleavage Karvelis, Tautvydas; Bigelyte, Greta; Young, Joshua K. Nucleic Acids Research https://doi.org/10.1101/654897	preprint	February 2020
Evaluating Protein Transfer Learning with TAPE Rao, Roshan; Bhattacharya, Nicholas; Thomas, Neil bioRxiv https://doi.org/10.1101/676825	preprint	June 2019
RNA-guided DNA insertion with CRISPR-associated transposases Strecker, Jonathan; Ladha, Alim; Gardner, Zachary Science, Vol. 365, Issue 6448 https://doi.org/10.1126/science.aax9181	journal	June 2019
Blood disease–causing and –suppressing transcriptional enhancers: general principles and GATA2 mechanisms Bresnick, Emery H.; Johnson, Kirby D. Blood Advances, Vol. 3, Issue 13 https://doi.org/10.1182/bloodadvances.2019000378	journal	July 2019
A catalog of CasX genome editing sites in common model organisms Roberson, Elisha D. O. BMC Genomics, Vol. 20, Issue 1 https://doi.org/10.1186/s12864-019-5924-6	journal	June 2019
Cardiac tissue engineering: state-of-the-art methods and outlook Nguyen, Anh H.; Marsh, Paul; Schmiess-Heine, Lauren Journal of Biological Engineering, Vol. 13, Issue 1 https://doi.org/10.1186/s13036-019-0185-0	journal	June 2019
Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency Tang, Xi-Dian; Gao, Fei; Liu, Ming-Jie Frontiers in Genetics, Vol. 10 https://doi.org/10.3389/fgene.2019.00551	journal	June 2019
A New Era of Prostate Cancer Precision Medicine Malik, Adil; Srinivasan, Srilakshmi; Batra, Jyotsna Frontiers in Oncology, Vol. 9 https://doi.org/10.3389/fonc.2019.01263	journal	November 2019
Epigenetic Control of a Local Chromatin Landscape Chiarella, Anna M.; Lu, Dongbo; Hathaway, Nathaniel A. International Journal of Molecular Sciences, Vol. 21, Issue 3 https://doi.org/10.3390/ijms21030943	journal	January 2020
Exploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era Shelake, Rahul Mahadev; Pramanik, Dibyajyoti; Kim, Jae-Yean Microorganisms, Vol. 7, Issue 8 https://doi.org/10.3390/microorganisms7080269	journal	August 2019
Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology Gale, Grant A. R.; Schiavon Osorio, Alejandra A.; Mills, Lauren A. Microorganisms, Vol. 7, Issue 10 https://doi.org/10.3390/microorganisms7100409	journal	September 2019
Evaluating Protein Transfer Learning with TAPE Rao, Roshan; Bhattacharya, Nicholas; Thomas, Neil arXiv https://doi.org/10.48550/arxiv.1906.08230	preprint	January 2019

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