Chapter 14
Genome Editing in Staphylococcus aureus by Conditional
Recombineering and CRISPR/Cas9-Mediated
Counterselection
Kelsi Penewit and Stephen J. Salipante
Abstract
Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered
experimental studies of this organism. We recently developed a system for recombineering and CRISPR/
Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA
oligonucleotides as substrates for introducing precise genomic modifications into the organism and for
performing lethal counterselection of unedited cells. These techniques make it possible to scalably and
inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and
detail general principles which are broadly applicable across a range of organisms for which equivalent
systems have been established.
Key words Electroporation, CRISPR, Cas9, Staphylococcus aureus, Genome editing, Recombineering, Genetic engineering, Isogenic strain
1 Introduction
Staphylococcus aureus is an important human and veterinary pathogen with a global distribution [1] that is responsible for a wide
variety of diseases [2–4]. However, it has proven difficult to genetically manipulate S. aureus, making it challenging to understand the
function of particular genes and pathways through the study of
isogenic strains. Consequently, key aspects of this organism’s biology remain incompletely understood [5].
Much progress has been made in developing methods and
techniques to introduce directed genetic changes into the
S. aureus genome, and multiple protocols now exist [6], each
having their own sets of benefits and drawbacks. Established
approaches share a common reliance on rare, homologous
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_14,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
127
Genome Editing in Staphylococcus aureus by Conditional
Recombineering and CRISPR/Cas9-Mediated
Counterselection
Kelsi Penewit and Stephen J. Salipante
Abstract
Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered
experimental studies of this organism. We recently developed a system for recombineering and CRISPR/
Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA
oligonucleotides as substrates for introducing precise genomic modifications into the organism and for
performing lethal counterselection of unedited cells. These techniques make it possible to scalably and
inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and
detail general principles which are broadly applicable across a range of organisms for which equivalent
systems have been established.
Key words Electroporation, CRISPR, Cas9, Staphylococcus aureus, Genome editing, Recombineering, Genetic engineering, Isogenic strain
1 Introduction
Staphylococcus aureus is an important human and veterinary pathogen with a global distribution [1] that is responsible for a wide
variety of diseases [2–4]. However, it has proven difficult to genetically manipulate S. aureus, making it challenging to understand the
function of particular genes and pathways through the study of
isogenic strains. Consequently, key aspects of this organism’s biology remain incompletely understood [5].
Much progress has been made in developing methods and
techniques to introduce directed genetic changes into the
S. aureus genome, and multiple protocols now exist [6], each
having their own sets of benefits and drawbacks. Established
approaches share a common reliance on rare, homologous
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_14,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
127