@inbook{1ed531988dc544b0a44713273b83554f,
title = "Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A",
abstract = "Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO2, C1 compounds (like methanol or methylated amines), or acetate as terminal electron acceptors. Methanogens are an untapped resource for biotechnological advances related to methane production as well as methane consumption. However, key biological features of these organisms remain poorly understood. One such feature is the enzyme methyl-coenzyme M reductase (referred to as MCR), which catalyzes the last step in the methanogenic pathway and results in methane formation. Gene essentiality has limited genetic analyses of MCR thus far. Therefore, studies of this important enzyme have been limited to biochemical and biophysical techniques that are especially laborious and often reliant on sophisticated instrumentation that is not commonly available. In this chapter, we outline our recently developed CRISPR–Cas9-based genome editing tools and describe how these tools have been used for the introduction of a tandem affinity purification tag at the chromosomal mcr locus in the model methanogen, Methanosarcina acetivorans C2A. We also report a protocol for rapid affinity purification of MCR from M. acetivorans C2A that will enable high-throughput studies of this enzyme in the future.",
keywords = "Cas9, Genome editing, Methane, Methanogens, Methanosarcina, Methyl-coenzyme M reductase",
author = "Nayak, {Dipti D.} and Metcalf, {William W.}",
note = "Funding Information: The authors acknowledge the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FG02-02ER15296 (to W.W.M.); the Carl R. Woese Institute for Genomic Biology postdoctoral fellowship (to D.D.N.); and the Simons Foundation Life Sciences Research Foundation postdoctoral fellowship (to D.D.N.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the funding sources. Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",
year = "2018",
doi = "10.1016/bs.mie.2018.10.012",
language = "English (US)",
isbn = "9780128163610",
series = "Methods in Enzymology",
publisher = "Academic Press Inc.",
pages = "325--347",
editor = "Fraser Armstrong",
booktitle = "Enzymes of Energy Technology",
address = "United States",
}