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Title: Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.
Authors:
ORCiD logo [1] ;  [1] ;  [1] ;  [2] ;  [1] ;  [1] ; ORCiD logo [3]
  1. Univ. of California, Berkeley, CA (United States)
  2. SLAC National Accelerator Lab., Menlo Park, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-76SF00515
Type:
Accepted Manuscript
Journal Name:
Nature Structural & Molecular Biology
Additional Journal Information:
Journal Volume: 24; Journal Issue: 10; Journal ID: ISSN 1545-9993
Publisher:
Nature Publishing Group
Research Org:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1426442

Knott, Gavin J., East-Seletsky, Alexandra, Cofsky, Joshua C., Holton, James M., Charles, Emeric, O'Connell, Mitchell R., and Doudna, Jennifer A.. Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme. United States: N. p., Web. doi:10.1038/nsmb.3466.
Knott, Gavin J., East-Seletsky, Alexandra, Cofsky, Joshua C., Holton, James M., Charles, Emeric, O'Connell, Mitchell R., & Doudna, Jennifer A.. Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme. United States. doi:10.1038/nsmb.3466.
Knott, Gavin J., East-Seletsky, Alexandra, Cofsky, Joshua C., Holton, James M., Charles, Emeric, O'Connell, Mitchell R., and Doudna, Jennifer A.. 2017. "Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme". United States. doi:10.1038/nsmb.3466. https://www.osti.gov/servlets/purl/1426442.
@article{osti_1426442,
title = {Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme},
author = {Knott, Gavin J. and East-Seletsky, Alexandra and Cofsky, Joshua C. and Holton, James M. and Charles, Emeric and O'Connell, Mitchell R. and Doudna, Jennifer A.},
abstractNote = {CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.},
doi = {10.1038/nsmb.3466},
journal = {Nature Structural & Molecular Biology},
number = 10,
volume = 24,
place = {United States},
year = {2017},
month = {9}
}