ArticleScientific reports2015
The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli.
Article in Scientific reports, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 134 papers.
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134 citing papers in PubMed, 262 citations in OpenAlex.
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- Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria.Nucleic acids research · 2026Article
- Bioengineering Interventions to Enhance the Capacity of the Gut Microbiota in Controlling Food Allergies.Life (Basel, Switzerland) · 2026Review
- Development and Optimization of CRISPR/Cas9-Assisted Recombineering inmicroPublication biology · 2026Article
- Chaperone dependency during biogenesis does not correlate with chaperone dependency during refolding.Molecular systems biology · 2026Article
- Using CRISPRi inJournal of microbiology & biology education · 2025Article
- Reprogramming the EnvZ-OmpR two-component system confers ethanol tolerance in Escherichia coli by stabilizing the outer membrane and altering ferric homeostasis.PLoS genetics · 2025Article
- RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.Communications biology · 2025Article
- High-throughput conjugation reveals strain specific recombination patterns enabling precise trait mapping in Escherichia coli.PLoS genetics · 2025Article
- Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.Biotechnology letters · 2025Article
- The RecBC complex protects single-stranded DNA gaps during lesion bypass.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency duringbioRxiv : the preprint server for biology · 2025Article
- In silico identification of gene targets to enhance C12 fatty acid production in Escherichia coli.Applied microbiology and biotechnology · 2025Article
- Heterologous synthesis of a simplified nitrogenase analog inScience advances · 2025Article
74 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.
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