ArticlePloS one2017
Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA expression.
Article in PloS one, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 47 papers.
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Who cites it
47 citing papers in PubMed.
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- Cell-type-specific RNA polymerase II activity maps in intact tissues provide a gateway to mammalian gene regulatory mechanisms in vivo.Developmental cell · 2026Article
- NUDT5 regulates purine metabolism and thiopurine sensitivity by interacting with PPAT.Science (New York, N.Y.) · 2025Article
- A next-generation dual guide CRISPR system for genetic interaction library screening.Nature communications · 2025Article
- Enhanced cleavage of genomicRNA biology · 2025Article
- Identification of a Distal Enhancer That Regulates TGF-β-Induced SNAI1 Expression.Cancer science · 2025Article
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- Development of multiplexed orthogonal base editor (MOBE) systems.Nature biotechnology · 2025Article
- Genetic regulation of TERT splicing affects cancer risk by altering cellular longevity and replicative potential.Nature communications · 2025Article
- Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.BMC genomics · 2025Article
- A scaleable inducible knockout system for studying essential gene function in the malaria parasite.Nucleic acids research · 2025Article
- Genetically edited human placental organoids cast new light on the role of ACE2.Cell death & disease · 2025Article
- An oligodendrocyte silencer element underlies the pathogenic impact of lamin B1 structural variants.Nature communications · 2025Article
- Protein Phosphatase 1 Regulatory Subunit PNUTS Prevents CENP-A Mislocalization and Chromosomal Instability.Molecular and cellular biology · 2025Article
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Authors and funding
3 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.
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