Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection

MBio
Kelsi PenewitStephen J Salipante

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 ex...Continue Reading

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Citations

Mar 15, 2019·PLoS Genetics·Mark ThomasVivek Iyer
Mar 25, 2019·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Gayetri Ramachandran, David Bikard
Apr 4, 2019·Microbiology·Christopher F SchusterAngelika Gründling
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Jun 6, 2020·ACS Synthetic Biology·Carlos Piñero-LambeaMaria Lluch-Senar
Oct 13, 2021·ACS Synthetic Biology·Lina J BirdSarah M Glaven

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Datasets Mentioned

BETA
PNPF00000000

Methods Mentioned

BETA
transgenic
targeted mutation
PCR

Software Mentioned

eBURST
MLST
IDT
CRISPRscan

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