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Title: RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

Abstract

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multi-subunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (50 tag) of the crRNA and the 30 flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csmmore » complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryoelectron microscopy and x-ray crystallography.« less

Authors:
ORCiD logo [1];  [2];  [3]
+ Show Author Affiliations
  1. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.
  2. Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
  3. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Univ. of California, Berkeley, CA (United States). Innovative Genomics Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). MBIB Division; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1627817
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics

Citation Formats

Liu, Tina Y., Iavarone, Anthony T., and Doudna, Jennifer A. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System. United States: N. p., 2017. Web. doi:10.1371/journal.pone.0170552.
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Liu, Tina Y., Iavarone, Anthony T., & Doudna, Jennifer A. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System. United States. https://doi.org/10.1371/journal.pone.0170552
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Liu, Tina Y., Iavarone, Anthony T., and Doudna, Jennifer A. Mon . "RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System". United States. https://doi.org/10.1371/journal.pone.0170552. https://www.osti.gov/servlets/purl/1627817.
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@article{osti_1627817,
title = {RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System},
author = {Liu, Tina Y. and Iavarone, Anthony T. and Doudna, Jennifer A.},
abstractNote = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multi-subunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (50 tag) of the crRNA and the 30 flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryoelectron microscopy and x-ray crystallography.},
doi = {10.1371/journal.pone.0170552},
journal = {PLoS ONE},
number = 1,
volume = 12,
place = {United States},
year = {Mon Jan 23 00:00:00 EST 2017},
month = {Mon Jan 23 00:00:00 EST 2017}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1371/journal.pone.0170552
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Figures / Tables:

Fig 1 Fig 1: Reconstitution and RNA cleavage activity of a T. thermophilus Csm complex (TthCsm) purified from E. coli with a defined crRNA species. (A) Components of the CRISPR locus and effector complexes of the T. thermophilus Type III-A Csm system. The complex is shown with 5 copies of Csm3 andmore » 4 copies of Csm2, but complexes with different numbers of these two subunits also exist. The CRISPR-4 locus associated with the system is shown (repeat is designated by R and spacer by S). The spacer 4.5 used for complex reconstitution encodes for one of the most abundant crRNAs found in the host organism. (B) Reconstitution and purification of TthCsm in E. coli. A plasmid containing genes encoding for Cas10/Csm1, and Csm2-5, with a His10 tag on Csm5, was co-transformed into E. coli with a plasmid containing genes for expression of T. thermophilus Cas6A and a single CRISPR array containing one copy of spacer 4.5. The purification steps are indicated. (C) TthCsm was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis following purification. Csm subunits are labeled, and a molecular weight ladder (M) is in the left lane (masses are given in kilodaltons). A GroEL contaminant (asterisk) was also identified by mass spectrometry (S2 Table). (D) TthCsm-mediated cleavage of a complementary (C) or noncomplementary (NC) 32P-labeled ssRNA oligonucleotide was tested in the presence of 2 mM MgCl2. Samples taken at 0, 5, 30, and 60 minutes after TthCsm addition were analyzed by denaturing PAGE. (E) Schematic of crRNA processing in Type III CRISPR-Cas systems is shown on the left. Pre-crRNAs are cleaved by Cas6 to generate an intermediate, which is then trimmed at the 3’-end, resulting in mature crRNAs. On the right, nucleic acids associated with the Csm complex were extracted and analyzed by denaturing PAGE. An ssDNA oligonucleotide ladder (M) was loaded in the right-most lane and nucleotide lengths are indicated.« less
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    Endogenous CRISPR-Cas System-Based Genome Editing and Antimicrobials: Review and Prospects
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