ArticleApplied and environmental microbiology2015
Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase.
Article in Applied and environmental microbiology, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 105 papers.
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Who cites it
105 citing papers in PubMed, 232 citations in OpenAlex.
- Molecular adaptations and engineering of extremophiles for synthetic biology and biotechnological applications.Frontiers in microbiology · 2026Review
- Integration-coupled activation of promoterless combinatorial pathway libraries inSynthetic biology (Oxford, England) · 2026Article
- Genome-scale metabolic modeling ofmSystems · 2025Article
- Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.Applied microbiology and biotechnology · 2025Article
- Genomic engineering inFrontiers in microbiology · 2025Article
- A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440.Microbial cell factories · 2024Article
- Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.Journal of fungi (Basel, Switzerland) · 2024Review
- Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus.Applied microbiology and biotechnology · 2024Article
- Enterococcus faecalis Strains with Compromised CRISPR-Cas Defense Emerge under Antibiotic Selection for a CRISPR-Targeted Plasmid.Applied and environmental microbiology · 2023Article
- CRISPR-Based Gene Editing inPharmaceuticals (Basel, Switzerland) · 2023Review
- Biotechnological and food synthetic biology potential of platform strain:Synthetic and systems biotechnology · 2023Review
- Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia.The ISME journal · 2023Article
- Optimized De Novo Eriodictyol Biosynthesis inInternational journal of molecular sciences · 2023Article
- Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens.Applied microbiology and biotechnology · 2023Article
- Tuning of Gene Expression inACS synthetic biology · 2022Article
- Evaluation of the immunogenicity of auxotrophicVirulence · 2022Article
- Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow.Biotechnology for biofuels and bioproducts · 2022Article
- Heterologous Gene Regulation in Clostridia: Rationally Designed Gene Regulation for Industrial and Medical Applications.ACS synthetic biology · 2022Article
- An efficient CRISPR/Cas9-based genome editing system for alkaliphilic Bacillus sp. N16-5 and application in engineering xylose utilization for D-lactic acid production.Microbial biotechnology · 2022Article
- Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.Journal of applied microbiology · 2022Review
45 more citing papers are in PubMed but not listed here.
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Authors and funding
9 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.
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