ArticleDNA research : an international journal for rapid publication of reports on genes and genomes2017
A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.
Article in DNA research : an international journal for rapid publication of reports on genes and genomes, 2017. 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.
- Article
- Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells.Nature communications · 2022Article
- BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors.Nature communications · 2021Article
- Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.Cell structure and function · 2021Article
- An efficient and precise method for generating knockout cell lines based on CRISPR-Cas9 system.Engineering in life sciences · 2020Article
- Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases.Nucleic acids research · 2020Article
- Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5'G-extended sgRNAs.Nature communications · 2020Article
- Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis.Microbial cell factories · 2019Article
- Ways of improving precise knock-in by genome-editing technologies.Human genetics · 2019Review
- Development of versatile non-homologous end joining-based knock-in module for genome editing.Scientific reports · 2018Article
- Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.Genome biology · 2017Article
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Authors and funding
17 authors.
Funding
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Abstract
The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self-cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.
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