Efficient and Scalable Precision Genome Editing in Staphylococcus aureus 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 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.

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

“…We obtained a recombination efficiency of ~5%, similar to that of studies in E. coli (Ellis et al 2001) and higher than previous λ Red Beta and RecT homologous systems found in other bacteria (van Kessel & Hatfull 2007;Yin et al 2015;Yang et al 2015;Swingle, Bao, et al 2010;Oh & Van Pijkeren 2014;Aparicio et al 2018;Penewit et al 2018;Lee et al 2017). In addition, W3 Beta was also functional in E. coli and performed at similar efficiencies to λ Red Beta.…”

“…Adapting recombineering for a new species is challenging; however, because the λ Red and RecET systems do not necessarily maintain high efficiency across different bacteria (Huang et al 2017;Hossain et al 2015;Hu et al 2014), there may be a dependence on host-specific machinery (Datta et al 2008;Muyrers et al 2000). Since recombineering was first applied in E. coli (Datsenko & Wanner 2000;Baba et al 2006), a few other phage homologous recombination systems have been found to promote recombination in Pseudomonas, Vibrio, Lactobacillus, Mycobacteria, Photorhabdus and Staphylococcus (van Kessel & Hatfull 2007;Yin et al 2015;Yang et al 2015;Swingle, Bao, et al 2010;Oh & Van Pijkeren 2014;Aparicio et al 2018;Penewit et al 2018;Lee et al 2017;Datta et al 2008), and we have explored whether this technology can be established in Shewanella. To maximize the recombination efficiency, we identified λ Red recombinase homologs in Shewanella species and have tested a system from Shewanella sp.…”

“…van Kessel & Hatfull 2007;Yin et al 2015;Yang et al 2015;Swingle, Bao, et al 2010;Oh & Van Pijkeren 2014;Aparicio et al 2018;Penewit et al 2018;Lee et al 2017), is a powerful tool for in vivo genome editing. In E. coli, recombineering has enabled targeted multiplexed editing strategies such as MAGE (Multiplex Automated Genome Engineering)(Wang et al 2009), TRMR (Trackable Multiplex Recombineering)(Warner et al 2010) and CREATE (CRISPR EnAbled Trackable genome Engineering)(Garst et al 2016), all which take advantage of massive DNA delivery by electroporation, highlighting the importance of this transformation method when performing recombineering experiments.…”

Artificial Selection for Pathogenicity Mutations in Staphylococcus aureus Identifies Novel Factors Relevant to Chronic Infection

“…It has been reported that the majority of mutations arising during in-host evolution in S. aureus inactivate protein function (34). To assess genes identified by our selection, we therefore initially generated isogenic single-gene nonpolar deletions using recombineering and CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated counterselection (63) or obtained bursa aurealis transposon mutants constructed in a different strain background (JE2) (64) when this strategy was unsuccessful. Two genes (mnhA and tufA) could not be constructed as nonpolar deletions, but strains were engineered to carry the specific missense mutations resulting from selection.…”

Malaria in Farmed Ungulates: an Exciting New System for Comparative Parasitology

“…A new paper by Guggisberg et al (7) now expands the observations of deer malaria in the United States by providing evidence for a high prevalence of these parasites in farmed white-tailed deer in Florida. The authors followed 33 fawns through their first year of life and observed that over 20% of them acquired infections of the hemosporidians.…”