RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease

Kazuki Kato; Sae Okazaki; Cian Schmitt-Ulms; Kaiyi Jiang; Wenyuan Zhou; Junichiro Ishikawa; Yukari Isayama; Shungo Adachi; Tomohiro Nishizawa; Kira S Makarova; Eugene V Koonin; Omar O Abudayyeh; Jonathan S Gootenberg; Hiroshi Nishimasu

doi:10.1126/science.add7347

RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease

Science. 2022 Nov 25;378(6622):882-889. doi: 10.1126/science.add7347. Epub 2022 Nov 3.

Authors

Kazuki Kato^#¹, Sae Okazaki^#¹, Cian Schmitt-Ulms^#², Kaiyi Jiang^#^{2

3}, Wenyuan Zhou², Junichiro Ishikawa¹, Yukari Isayama¹, Shungo Adachi⁴, Tomohiro Nishizawa⁵, Kira S Makarova⁶, Eugene V Koonin⁶, Omar O Abudayyeh², Jonathan S Gootenberg², Hiroshi Nishimasu^{1

7

8

9}

Affiliations

¹ Structural Biology Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
² McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁴ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
⁵ Graduate School of Medical Life Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa 236-0027, Japan.
⁶ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
⁷ Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
⁸ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁹ Inamori Research Institute for Science, 620 Suiginya-cho, Shimogyo-ku, Kyoto 600-8411, Japan.

^# Contributed equally.

PMID: 36423304
DOI: 10.1126/science.add7347

Abstract

The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.

Publication types

Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Proteins* / chemistry
CRISPR-Associated Proteins* / metabolism
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats*
Cryoelectron Microscopy
Deltaproteobacteria* / enzymology
Endonucleases* / chemistry
Endonucleases* / metabolism
HEK293 Cells
Protein Conformation
Proteolysis*
RNA, Guide, CRISPR-Cas Systems* / chemistry
RNA, Guide, CRISPR-Cas Systems* / metabolism

Substances

Endonucleases
CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems

Abstract

Publication types

MeSH terms

Substances

Grants and funding