ArticlePloS one2020
An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.
Article in PloS one, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
16 citing papers in PubMed, 31 citations in OpenAlex.
- Design and transfection of CRISPR/Cas9 constructs for the myostatin gene in Labeo rohita muscle cells.Scientific reports · 2026Article
- CRISPR-driven diagnostics: Molecular mechanisms, clinical efficacy and translational challenges.Clinical and translational medicine · 2025Review
- Article
- Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication.Autophagy · 2024Article
- Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.ACS pharmacology & translational science · 2024Article
- Review
- Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.Scientific reports · 2023Article
- NOG1 downregulates type I interferon production by targeting phosphorylated interferon regulatory factor 3.PLoS pathogens · 2023Article
- Picornavirus infection enhances aspartate by the SLC38A8 transporter to promote viral replication.PLoS pathogens · 2023Article
- Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.PLoS pathogens · 2023Article
- Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.Cancer cell international · 2022Article
- Fast-Track and Integration-Free Method of Genome Editing by CRISPR/Cas9 in Murine Pluripotent Stem Cells.Frontiers in cell and developmental biology · 2022Article
- HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation.Life (Basel, Switzerland) · 2021Article
- A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries.Nucleic acids research · 2021Article
- FOLFOX Therapy Induces Feedback Upregulation of CD44v6 through YB-1 to Maintain Stemness in Colon Initiating Cells.International journal of molecular sciences · 2021Article
- Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells.Animals : an open access journal from MDPI · 2020Article
Corrections and comments
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Authors and funding
11 authors at 1 institution in 1 country.
Funding
Abstract
A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.