Erratum for Penewit et al., “Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection”

Penewit, Kelsi; Holmes, Elizabeth A.; McLean, Kathryn; Ren, Mingxin; Waalkes, Adam; Salipante, Stephen J.

doi:10.1128/mbio.01839-18

Cited by 9 publications

(10 citation statements)

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“…Although RecT homologs have demonstrated more cross‐species activity, RecE appears to show more limited activity in an evolutionarily distant species . The Rpsl and RpoB mutagenesis‐based evaluation workflow facilitates the activity assessment of various RecTs, which will contribute to RecT‐assisted ssDNA recombineering‐related genetic engineering and, additionally, enrich the library of molecular genetic elements for Lactobacillus genome editing. However, a genome‐wide survey of RecE/T association will still be challenging for some species without an endogenous phage.…”

Section: Discussionmentioning

confidence: 99%

Development of a RecE/T‐Assisted CRISPR–Cas9 Toolbox for Lactobacillus

Huang

Song

Yang

2019

Biotechnology Journal

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Lactobacilli are members of a large family involved in industrial food fermentation, therapeutics, and health promotion. However, the development of genetic manipulation tools for this genus lags behind its relative industrial and medical significance. The development of clustered regularly interspaced short palindromic repeat (CRISPR)‐based genome engineering for Lactobacillus is now underway. However, some Lactobacillus species are sensitive to CRISPR‐Cas9 induced double strand breaks (DSBs) due to a deficiency in homology‐directed repair (HDR), which allows chromosomal genetic editing. Here, phage‐derived RecE/T is coupled with CRISPR–Cas9 and the transcriptional activity of broad‐spectrum host promoters is assessed to set up a versatile toolbox containing a recombination helper plasmid and a broad host CRISPR–Cas9 editing plasmid, which enables efficient genome editing in Lactobacillus plantarum (L. plantarum) WCFS1 and Lactobacillus brevis (L. brevis) ATCC367. The RecE/T‐assisted CRISPR–Cas9 toolbox realizes single gene deletions at an efficiency of 50–100% in seven days. Furthermore, the chromosomal gene replacement of Lp_0537 using a P23‐pyruvate decarboxylase (pdc) expression cassette is accomplished with an efficiency of 35.7%. This study establises a RecE/T‐assisted CRISPR genome editing toolbox for L. plantarum WCFS1 and L. brevis ATCC367 and also demonstrate that RecE/T‐assisted CRISPR–Cas9 is an effective genome editing system, which can be readily implemented in Lactobacilli.

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Section: Discussionmentioning

confidence: 99%

Development of a RecE/T‐Assisted CRISPR–Cas9 Toolbox for Lactobacillus

Huang

Song

Yang

2019

Biotechnology Journal

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“…When using synthetic single‐strand oligonucleotides as a repair template, the auxiliary recombination system can be simplified, utilizing the SSAP only (Table S1, Supporting Information). Studies have indicated that λ Bet is not a versatile SSAP, which only retains function in some Proteobacteria species . In contrast, RecT efficiently mediates recombination in some gram‐positive bacteria .…”

Section: Repair Of Dna Double‐strand Breakmentioning

confidence: 99%

“…Intriguingly, although they function similarly and share close quaternary structure, their sequence similarities are poor, indicating that they may evolve independently but convergently, posing a challenge in identifying new SSAPs for CRISPR‐based genome editing. A recent study had screened an array of SSAPs to identify one for establishing a CRISPR‐based genome editing method in S. aureus , which allows the usage of short ssDNA as a repair template . The authors found five SSAPs, including λ Bet, three putative SSAPs originated from S. aureus , and a predicated SSAP from Legionella pneumophilia , barely worked in S. aureus , whereas only one SSAP, termed EF2132, derived from Enterococcus faecalis , efficiently rescued the bacteria from lethal genome targeting in S. aureus using short ss oligos as repair templates.…”

Section: Repair Of Dna Double‐strand Breakmentioning

confidence: 99%

“…Studies have indicated that λ Bet is not a versatile SSAP, which only retains function in some Proteobacteria species. [126,127] In contrast, RecT efficiently mediates recombination in some gram-positive bacteria. [16,67,102,128,129] Previous bioinformatics study has classified SSAP in three distinct superfamilies distributed in most organisms.…”

Section: Complementing Auxiliary Recombination Systemsmentioning

confidence: 99%

“…A recent study had screened an array of SSAPs to identify one for establishing a CRISPR-based genome editing method in S. aureus, which allows the usage of short ssDNA as a repair template. [127] The authors found five SSAPs, including λ Bet, three putative SSAPs originated from S. aureus, and a predicated SSAP from Legionella pneumophilia, barely worked in S. aureus, whereas only one SSAP, termed EF2132, derived from Enterococcus faecalis, efficiently rescued the bacteria from lethal genome targeting in S. aureus using short ss oligos as repair templates. Another study had identified a new homologue of λ Bet in a prophage from P. putida DOT-T1E strain, termed Ssr, which efficiently mediated the invasion to replication fork by singlestrand oligonucleotides.…”

Section: Complementing Auxiliary Recombination Systemsmentioning

confidence: 99%

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Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Wang

Zhang

et al. 2019

Small Methods

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Gene editing is a fundamental technique for the identification of the linkages from genetic determinants to significant biological phenotypes, and the engineering of industrial strains to produce fine chemicals. Recently, the primitive bacterial immunity systems, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas systems, have been engineered as programmable nucleases to deliver double‐strand breaks (DSBs) at user‐defined loci. The DSB is repaired via either homology‐direct repair or the non‐homologous end joining pathway, generating a mutant in various organisms, and leading a revolution in the field of gene editing. This paper reviews the structural and molecular details of CRISPR‐Cas systems, describing their applications and challenges of genome targeting in bacterial cells. Moreover, the DSB repair pathways in bacteria are summarized and a guideline for selecting repair machines and maximizing the recombination efficiency is presented. In addition, the plasmid curing approaches in bacteria are outlined for iterative gene editing or downstream biological applications. Furthermore, CRISPR‐based transcriptional regulation, base editing, and transposition are also introduced. Finally, this paper prospects the future directions for improving CRISPR‐based gene editing methods in bacteria.

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Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva

Zheng

Wang

Chen

et al. 2021

Biotechnology Journal

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Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01‐1 is a broad‐spectrum antagonistic bacterium present in plants. However, no natural products have been isolated from this strain till date. Corresponding biosynthetic gene clusters to natural products is an effective method for bioprospecting, for which, genome manipulation tools are essential. We previously developed Pseudomonas‐specific phage‐derived homologous recombination systems for genetic engineering in four Pseudomonas species. Herein, we report the application of these recombineering systems in Pseudomonas parafulva CRS01‐1, along with structural elucidation and bioactivity evaluation of natural products. The Pseudomonas recombineering toolbox established before in different four species is efficient for genome mining and bioactive metabolite discovery from more distant species.

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Erratum for Penewit et al., “Efficient and Scalable Precision Genome Editing in Staphylococcus aureus through Conditional Recombineering and CRISPR/Cas9-Mediated Counterselection”

Abstract: The first name of the third author was misspelled. The name should appear as shown above.

Cited by 9 publications

References 0 publications

Development of a RecE/T‐Assisted CRISPR–Cas9 Toolbox for Lactobacillus

Development of a RecE/T‐Assisted CRISPR–Cas9 Toolbox for Lactobacillus

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva

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