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Your search keyword '"John J Desmarais"' showing total 3 results
Start Over Author "John J Desmarais" Topic crispr-cas systems
3 results on '"John J Desmarais"'

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

Author

Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brötzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, and Doudna JA

Subjects
CRISPR-Associated Proteins chemistry, CRISPR-Associated Proteins metabolism, Cryoelectron Microscopy, DNA chemistry, DNA metabolism, DNA ultrastructure, DNA Cleavage, Escherichia coli genetics, Evolution, Molecular, Gene Silencing, Genome, Bacterial genetics, Genome, Human genetics, Humans, Models, Molecular, Nucleic Acid Conformation, Protein Domains, RNA, Guide, CRISPR-Cas Systems metabolism, CRISPR-Associated Proteins classification, CRISPR-Associated Proteins ultrastructure, CRISPR-Cas Systems genetics, Gene Editing
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.

Published
2019
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2. Accelerated RNA detection using tandem CRISPR nucleases

Author

Chunyu Zhao, Abdul Bhuiya, Jennifer A. Doudna, Melanie Ott, Emeric J Charles, Ming X. Tan, Brittney W. Thornton, Patrick D. Hsu, Amanda Mok, Arturo M. Escajeda, María Díaz de León Derby, Katherine S. Pollard, Daniel A. Fletcher, David F. Savage, G. Renuka Kumar, Noam Prywes, Tina Y. Liu, Dylan C. J. Smock, Shineui Kim, John J Desmarais, Eli J Dugan, Parinaz Fozouni, Sungmin Son, Liana F. Lareau, Neil A. Switz, Andrew R. Harris, Stephanie I. Stephens, Maxim Armstrong, Jeffrey Shu, Shrutee Jakhanwal, Anthony T. Iavarone, Roger McIntosh, Shreeya Agrawal, and Gavin J. Knott

Subjects
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Microfluidics, RNA-binding protein, Quantitative Reverse Transcriptase PCR, RNA-binding proteins, Computational biology, Article, CRISPR, Humans, Molecular Biology, Nuclease, Fluorescent reporter, biology, Tandem, Chemistry, Activator (genetics), Reverse Transcriptase Polymerase Chain Reaction, SARS-CoV-2, RNA, COVID-19, Cell Biology, Molecular diagnostics, Publisher Correction, biology.protein, RNA, Viral, Infectious diseases, CRISPR-Cas Systems, Chemical tools
Abstract

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

Published
2021

3. CRISPR-CasX is an RNA-dominated enzyme active for human genome editing

Author

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

Subjects
0301 basic medicine, Models, Molecular, CRISPR-Associated Proteins, Computational biology, Biology, Genome, Article, Evolution, Molecular, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Genome editing, Protein Domains, Escherichia coli, Humans, Clustered Regularly Interspaced Short Palindromic Repeats, Guide RNA, Gene Silencing, DNA Cleavage, Gene Editing, Multidisciplinary, Cas9, Genome, Human, Cryoelectron Microscopy, RNA, DNA, 030104 developmental biology, chemistry, Nucleic acid, Nucleic Acid Conformation, Human genome, CRISPR-Cas Systems, 030217 neurology & neurosurgery, Genome, Bacterial, RNA, Guide, Kinetoplastida
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.

Published
2019

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