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Title: Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Abstract

Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeonMethanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon,Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length,more » most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.« less

Authors:
ORCiD logo; ORCiD logo
Publication Date:
Research Org.:
Univ. of Illinois at Urbana–Champaign, Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Chemical Sciences, Geosciences & Biosciences Division
OSTI Identifier:
1345844
Alternate Identifier(s):
OSTI ID: 1465485; OSTI ID: 1595333
Grant/Contract Number:  
FG02-02ER15296
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 114 Journal Issue: 11; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Cas9; Archaea; methanogens; Methanosarcina; genetics

Citation Formats

Nayak, Dipti D., and Metcalf, William W. Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans. United States: N. p., 2017. Web. doi:10.1073/pnas.1618596114.
Nayak, Dipti D., & Metcalf, William W. Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans. United States. doi:10.1073/pnas.1618596114.
Nayak, Dipti D., and Metcalf, William W. Mon . "Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans". United States. doi:10.1073/pnas.1618596114.
@article{osti_1345844,
title = {Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans},
author = {Nayak, Dipti D. and Metcalf, William W.},
abstractNote = {Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeonMethanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon,Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.},
doi = {10.1073/pnas.1618596114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 11,
volume = 114,
place = {United States},
year = {2017},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1073/pnas.1618596114

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Cited by: 5 works
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Works referenced in this record:

An updated evolutionary classification of CRISPR–Cas systems
journal, September 2015

  • Makarova, Kira S.; Wolf, Yuri I.; Alkhnbashi, Omer S.
  • Nature Reviews Microbiology, Vol. 13, Issue 11, p. 722-736
  • DOI: 10.1038/nrmicro3569

Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea
journal, March 2004


Genetic manipulation of Methanosarcina spp.
journal, January 2012


Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit
journal, July 2016


Molecular, genetic, and biochemical characterization of the serC gene of Methanosarcina barkeri Fusaro.
journal, October 1996


Homologous recombination in the archaea: the means justify the ends
journal, January 2011

  • White, Malcolm F.
  • Biochemical Society Transactions, Vol. 39, Issue 1
  • DOI: 10.1042/BST0390015

The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014


Archaea in Biogeochemical Cycles
journal, September 2013


Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq
journal, January 2014

  • Barrick, Jeffrey E.; Colburn, Geoffrey; Deatherage, Daniel E.
  • BMC Genomics, Vol. 15, Issue 1
  • DOI: 10.1186/1471-2164-15-1039

Environmental Diversity of Bacteria and Archaea
journal, August 2001


Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase
journal, February 2010


The Genome of M. acetivorans Reveals Extensive Metabolic and Physiological Diversity
journal, April 2002


A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


PROCESSING OF RECOMBINATION INTERMEDIATES BY THE R uv ABC PROTEINS
journal, December 1997


Metabolic versatility in methanogens
journal, October 2014


Molecular basis for DNA strand displacement by NHEJ repair polymerases
journal, September 2015

  • Bartlett, Edward J.; Brissett, Nigel C.; Plocinski, Przemyslaw
  • Nucleic Acids Research, Vol. 44, Issue 5
  • DOI: 10.1093/nar/gkv965

Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species
journal, June 2016


Metabolic, Phylogenetic, and Ecological Diversity of the Methanogenic Archaea
journal, March 2008

  • Liu, Yuchen; Whitman, William B.
  • Annals of the New York Academy of Sciences, Vol. 1125, Issue 1
  • DOI: 10.1196/annals.1419.019

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates
journal, May 2013

  • Bartlett, E. J.; Brissett, N. C.; Doherty, A. J.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 22
  • DOI: 10.1073/pnas.1302616110

Pyrrolysine Encoded by UAG in Archaea: Charging of a UAG-Decoding Specialized tRNA
journal, May 2002


Harnessing Type I and Type III CRISPR-Cas systems for genome editing
journal, October 2015

  • Li, Yingjun; Pan, Saifu; Zhang, Yan
  • Nucleic Acids Research, Vol. 44, Issue 4
  • DOI: 10.1093/nar/gkv1044

CRISPR/Cas, the Immune System of Bacteria and Archaea
journal, January 2010


Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment
journal, March 2015

  • Youngblut, Nicholas D.; Wirth, Joseph S.; Henriksen, James R.
  • The ISME Journal, Vol. 9, Issue 10
  • DOI: 10.1038/ismej.2015.31

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production
journal, November 2016


Genome engineering using the CRISPR-Cas9 system
journal, October 2013


Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species
journal, May 2012

  • Kim, Seung Young; Ju, Kou-San; Metcalf, William W.
  • Antimicrobial Agents and Chemotherapy, Vol. 56, Issue 8
  • DOI: 10.1128/AAC.06478-11

Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya.
journal, June 1990

  • Woese, C. R.; Kandler, O.; Wheelis, M. L.
  • Proceedings of the National Academy of Sciences, Vol. 87, Issue 12
  • DOI: 10.1073/pnas.87.12.4576

Consequences of Cas9 cleavage in the chromosome of Escherichia coli
journal, April 2016

  • Cui, Lun; Bikard, David
  • Nucleic Acids Research, Vol. 44, Issue 9
  • DOI: 10.1093/nar/gkw223

Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity
journal, January 1993


Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014


High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
journal, June 2013

  • Fu, Yanfang; Foden, Jennifer A.; Khayter, Cyd
  • Nature Biotechnology, Vol. 31, Issue 9
  • DOI: 10.1038/nbt.2623

Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems
journal, January 2014


Mycobacterial Ku and Ligase Proteins Constitute a Two-Component NHEJ Repair Machine
journal, October 2004


An Anaerobic, Intrachamber Incubator for Growth ofMethanosarcina spp. on Methanol-Containing Solid Media
journal, January 1998

  • Metcalf, William W.; Zhang, Jun Kai; Wolfe, Ralph S.
  • Applied and Environmental Microbiology, Vol. 64, Issue 2
  • DOI: 10.1128/AEM.64.2.768-770.1998

Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

A genetic system for Archaea of the genus Methanosarcina: Liposome-mediated transformation and construction of shuttle vectors
journal, March 1997

  • Metcalf, W. W.; Zhang, J. K.; Apolinario, E.
  • Proceedings of the National Academy of Sciences, Vol. 94, Issue 6
  • DOI: 10.1073/pnas.94.6.2626

Exploiting CRISPR–Cas immune systems for genome editing in bacteria
journal, February 2016


Genetic Screens in Human Cells Using the CRISPR-Cas9 System
journal, December 2013


Current and future prospects for CRISPR-based tools in bacteria: CRISPR-Based Tools in Bacteria
journal, October 2015

  • Luo, Michelle L.; Leenay, Ryan T.; Beisel, Chase L.
  • Biotechnology and Bioengineering, Vol. 113, Issue 5
  • DOI: 10.1002/bit.25851

    Works referencing / citing this record:

    Building a genome engineering toolbox in nonmodel prokaryotic microbes
    journal, May 2018

    • Freed, Emily; Fenster, Jacob; Smolinski, Sharon L.
    • Biotechnology and Bioengineering, Vol. 115, Issue 9
    • DOI: 10.1002/bit.26727

    Molecular underpinnings for microbial extracellular electron transfer during biogeochemical cycling of earth elements
    journal, March 2019


    A biochemical framework for anaerobic oxidation of methane driven by Fe(III)-dependent respiration
    journal, April 2018


    Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance
    journal, June 2018


    An evolving view of methane metabolism in the Archaea
    journal, January 2019


    Common principles and best practices for engineering microbiomes
    journal, September 2019

    • Lawson, Christopher E.; Harcombe, William R.; Hatzenpichler, Roland
    • Nature Reviews Microbiology, Vol. 17, Issue 12
    • DOI: 10.1038/s41579-019-0255-9

    Making quantitative sense of electromicrobial production
    journal, May 2019

    • Claassens, Nico J.; Cotton, Charles A. R.; Kopljar, Dennis
    • Nature Catalysis, Vol. 2, Issue 5
    • DOI: 10.1038/s41929-019-0272-0

    Editing streptomycete genomes in the CRISPR/Cas9 age
    journal, January 2019

    • Alberti, Fabrizio; Corre, Christophe
    • Natural Product Reports, Vol. 36, Issue 9
    • DOI: 10.1039/c8np00081f

    Multiplex genome editing of microorganisms using CRISPR-Cas
    journal, April 2019

    • Adiego-Pérez, Belén; Randazzo, Paola; Daran, Jean Marc
    • FEMS Microbiology Letters, Vol. 366, Issue 8
    • DOI: 10.1093/femsle/fnz086

    The streptothricin acetyltransferase (sat) gene as a positive selectable marker for methanogenic archaea
    journal, September 2019

    • Farley, Kristen R.; Metcalf, William W.
    • FEMS Microbiology Letters, Vol. 366, Issue 17
    • DOI: 10.1093/femsle/fnz216

    CRISPR-Cas12a-Assisted Recombineering in Bacteria
    journal, June 2017

    • Yan, Mei-Yi; Yan, Hai-Qin; Ren, Gai-Xian
    • Applied and Environmental Microbiology, Vol. 83, Issue 17
    • DOI: 10.1128/aem.00947-17

    History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology
    journal, January 2018

    • Ishino, Yoshizumi; Krupovic, Mart; Forterre, Patrick
    • Journal of Bacteriology, Vol. 200, Issue 7
    • DOI: 10.1128/jb.00580-17

    Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci
    journal, March 2019


    Methanogens: biochemical background and biotechnological applications
    journal, January 2018


    A Genetic System for Methanocaldococcus jannaschii: An Evolutionary Deeply Rooted Hyperthermophilic Methanarchaeon
    journal, July 2019

    • Susanti, Dwi; Frazier, Mary C.; Mukhopadhyay, Biswarup
    • Frontiers in Microbiology, Vol. 10
    • DOI: 10.3389/fmicb.2019.01256