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Title: Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [4] ;  [1] ;  [5] ;  [6] ;  [7] ;  [3] ;  [6] ;  [2] ;  [8] ;  [1]
  1. Rutgers Univ., Piscataway, NJ (United States)
  2. Univ. of Toronto, ON (Canada)
  3. Montana State Univ., Bozeman, MT (United States)
  4. Purdue Univ., West Lafayette, IN (United States)
  5. Russian Academy of Sciences (RAS), Moscow (Russian Federation); Skolkovo Inst. of Science and Technology (Russian Federation)
  6. Johns Hopkins Univ., Baltimore, MD (United States)
  7. Skolkovo Inst. of Science and Technology (Russian Federation); National Library of Medicine (NLM), Bethesda, MD (United States)
  8. Skolkovo Inst. of Science and Technology (Russian Federation); Rutgers Univ., Piscataway, NJ (United States); Russian Academy of Sciences (RAS), Moscow (Russian Federation); St. Petersburg State Polytechnic Univ. (Russian Federation)
Publication Date:
Grant/Contract Number:
SC0012518
Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 44; Journal Issue: 22; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Research Org:
State University of New Jersey, Piscataway, NJ, USA
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1425391

Kuznedelov, Konstantin, Mekler, Vladimir, Lemak, Sofia, Tokmina-Lukaszewska, Monika, Datsenko, Kirill A., Jain, Ishita, Savitskaya, Ekaterina, Mallon, John, Shmakov, Sergey, Bothner, Brian, Bailey, Scott, Yakunin, Alexander F., Severinov, Konstantin, and Semenova, Ekaterina. Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. United States: N. p., Web. doi:10.1093/nar/gkw914.
Kuznedelov, Konstantin, Mekler, Vladimir, Lemak, Sofia, Tokmina-Lukaszewska, Monika, Datsenko, Kirill A., Jain, Ishita, Savitskaya, Ekaterina, Mallon, John, Shmakov, Sergey, Bothner, Brian, Bailey, Scott, Yakunin, Alexander F., Severinov, Konstantin, & Semenova, Ekaterina. Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation. United States. doi:10.1093/nar/gkw914.
Kuznedelov, Konstantin, Mekler, Vladimir, Lemak, Sofia, Tokmina-Lukaszewska, Monika, Datsenko, Kirill A., Jain, Ishita, Savitskaya, Ekaterina, Mallon, John, Shmakov, Sergey, Bothner, Brian, Bailey, Scott, Yakunin, Alexander F., Severinov, Konstantin, and Semenova, Ekaterina. 2016. "Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation". United States. doi:10.1093/nar/gkw914. https://www.osti.gov/servlets/purl/1425391.
@article{osti_1425391,
title = {Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation},
author = {Kuznedelov, Konstantin and Mekler, Vladimir and Lemak, Sofia and Tokmina-Lukaszewska, Monika and Datsenko, Kirill A. and Jain, Ishita and Savitskaya, Ekaterina and Mallon, John and Shmakov, Sergey and Bothner, Brian and Bailey, Scott and Yakunin, Alexander F. and Severinov, Konstantin and Semenova, Ekaterina},
abstractNote = {The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo. Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multi-subunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.},
doi = {10.1093/nar/gkw914},
journal = {Nucleic Acids Research},
number = 22,
volume = 44,
place = {United States},
year = {2016},
month = {10}
}

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