ArticleMethods (San Diego, Calif.)2019
A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.
Article in Methods (San Diego, Calif.), 2019. 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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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, 24 citations in OpenAlex.
- CRISPR-Edited Cell Lines: A New Era in Functional Oncology Research.Current pharmaceutical design · 2026Review
- Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.Theranostics · 2026Review
- Mammalian antiviral proteins ZAP and KHNYN can independently restrict CpG-enriched avian viruses.PLoS biology · 2025Article
- Mammalian ZAP and KHNYN can independently restrict CpG-enriched avian viruses.bioRxiv : the preprint server for biology · 2025Article
- CRISPR/Cas9 as a Mutagenic Factor.International journal of molecular sciences · 2024Review
- Mitochondrial double-stranded RNA triggers induction of the antiviral DNA deaminase APOBEC3A and nuclear DNA damage.The Journal of biological chemistry · 2023Article
- Article
- APOBEC3 degradation is the primary function of HIV-1 Vif for virus replication in the myeloid cell line THP-1.bioRxiv : the preprint server for biology · 2023Article
- Small Molecule Inhibitors of Activation-Induced Deaminase Decrease Class Switch Recombination in B Cells.ACS pharmacology & translational science · 2021Article
- Article
- The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.Clinical cancer research : an official journal of the American Association for Cancer Research · 2020Article
- MagnEdit-interacting factors that recruit DNA-editing enzymes to single base targets.Life science alliance · 2020Article
- The deaminase APOBEC3B triggers the death of cells lacking uracil DNA glycosylase.Proceedings of the National Academy of Sciences of the United States of America · 2019Article
- A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.Antibodies (Basel, Switzerland) · 2019Article
- The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1.The Journal of biological chemistry · 2019Article
- HIV-1 restriction by endogenous APOBEC3G in the myeloid cell line THP-1.The Journal of general virology · 2019Article
Corrections and comments
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Authors and funding
5 authors at 3 institutions in 1 country.
Funding
Abstract
A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.
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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.