Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection

ABSTRACT Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using s...

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Autores principales:	Kelsi Penewit, Elizabeth A. Holmes, Kathryn McLean, Mingxin Ren, Adam Waalkes, Stephen J. Salipante
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR Cas9 Staphylococcus aureus genetic engineering genome editing mutS Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bc80c69d71cf43f2813b54ad9b9b729a
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spelling	oai:doaj.org-article:bc80c69d71cf43f2813b54ad9b9b729a2021-11-15T15:53:25ZEfficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection10.1128/mBio.00067-182150-7511https://doaj.org/article/bc80c69d71cf43f2813b54ad9b9b729a2018-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00067-18https://doaj.org/toc/2150-7511ABSTRACT Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds. IMPORTANCE Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus. In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureus genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.Kelsi PenewitElizabeth A. HolmesKathryn McLeanMingxin RenAdam WaalkesStephen J. SalipanteAmerican Society for MicrobiologyarticleCRISPRCas9Staphylococcus aureusgenetic engineeringgenome editingmutSMicrobiologyQR1-502ENmBio, Vol 9, Iss 1 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR Cas9 Staphylococcus aureus genetic engineering genome editing mutS Microbiology QR1-502
spellingShingle	CRISPR Cas9 Staphylococcus aureus genetic engineering genome editing mutS Microbiology QR1-502 Kelsi Penewit Elizabeth A. Holmes Kathryn McLean Mingxin Ren Adam Waalkes Stephen J. Salipante Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
description	ABSTRACT Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds. IMPORTANCE Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus. In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureus genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.
format	article
author	Kelsi Penewit Elizabeth A. Holmes Kathryn McLean Mingxin Ren Adam Waalkes Stephen J. Salipante
author_facet	Kelsi Penewit Elizabeth A. Holmes Kathryn McLean Mingxin Ren Adam Waalkes Stephen J. Salipante
author_sort	Kelsi Penewit
title	Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
title_short	Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
title_full	Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
title_fullStr	Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
title_full_unstemmed	Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection
title_sort	efficient and scalable precision genome editing in <italic toggle="yes">staphylococcus aureus</italic> through conditional recombineering and crispr/cas9-mediated counterselection
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/bc80c69d71cf43f2813b54ad9b9b729a
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Efficient and Scalable Precision Genome Editing in <italic toggle="yes">Staphylococcus aureus</italic> through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection

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