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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 archaeon Methanosarcina 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 kbmore » in length, most of which appear to have occurred at regions of naturally occurring microhomology. In conclusion, 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 [1]; ORCiD logo [1]
  1. Univ. of Illinois at Urbana–Champaign, Urbana, IL (United States)
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)
OSTI Identifier:
1345844
Alternate Identifier(s):
OSTI ID: 1465485
Grant/Contract Number:  
FG02-02ER15296
Resource Type:
Journal Article: Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 11; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
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 archaeon Methanosarcina 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. In conclusion, 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},
issn = {0027-8424},
number = 11,
volume = 114,
place = {United States},
year = {2017},
month = {3}
}

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

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Cited by: 5 works
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Works referenced in this record:

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