ArticleMicrobial cell factories2020
Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli.
Article in Microbial cell factories, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 19 citations in OpenAlex.
- Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.Archives of microbiology · 2026Review
- Probing the limits of genetic recoding using multi-omics-guided evolution.Nature communications · 2026Article
- Multiplex gene editing drives revolution in crop breeding: overlaid editing of multiple genes and customization of complex traits.Advanced biotechnology · 2026Review
- Genomic medicine in hepatology: mechanisms and liver treatment strategies.Molecular medicine (Cambridge, Mass.) · 2025Review
- Synthetic genomes unveil the effects of synonymous recoding.bioRxiv : the preprint server for biology · 2024Article
- Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.Microbial cell factories · 2022Article
- Advances in CRISPR/Cas9.BioMed research international · 2022Review
- Recombineering and MAGE.Nature reviews. Methods primers · 2021Article
- Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement.Frontiers in plant science · 2021Review
- Pruning and Tending Immune Memories: Spacer Dynamics in the CRISPR Array.Frontiers in microbiology · 2021Review
- MultiplexedViruses · 2020Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
Abstract
backgroundCo-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system.
resultsTo address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli.
conclusionsThe RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.
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