Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression

Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Nakatani, Kiyoharu; Takayama, Kazuo; Tachibana, Masashi; Mizuguchi, Hiroyuki

doi:10.1248/bpb.b16-00700

Cited by 7 publications

(10 citation statements)

References 26 publications

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“…The cell growth arrest and apoptosis upon CRISPR-Cas complex delivery has also been suggested to result from type I interferon (IFN) response, which can be activated upon plasmid, protein and lentiviral transfection 2,14,15 . Since the Toll-like receptor, Interferon-α and Interleukin 1β signaling comprise the major branches of this pathway, we tested the effect of several established IFN inhibitors on precision genome editing using the RPE-1-GFP cell line described above ( Supplementary Table S2).…”

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confidence: 99%

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Haapaniemi

Botla

Persson

et al. 2017

Preprint

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We report here that genome editing by CRISPR/Cas9 induces a p53-mediated DNA damage response and cell cycle arrest. Transient inhibition of p53 prevents this response, and increases the rate of homologous recombination more than five-fold. This provides a way to improve precision genome editing of normal cells, but warrants caution in using CRISPR for human therapies until the mechanism of the activation of p53 is elucidated.

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confidence: 99%

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Haapaniemi

Botla

Persson

et al. 2017

Preprint

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“…pHM-U6-asgRNA-AAVS1, pHM-U6-lbgRNA-AAVS1 and pHM-U6-cas9gRNA-AAVS1 were generated by insertion of U6-asgRNA-AAVS1, U6-lbgRNA-AAVS1 and U6-cas9gRNA-AAVS1 fragments from pU6-asgRNA-AAVS1, pU6-lbgRNA-AAVS1 and pX330-AAVS1 16) into pHM5 shuttle vector, 17) respectively. The reporter plasmid pCAG-EGxxFP-AAVS1 18) containing approximately 500 bp long genomic sequence of AAVS1 locus and the control plasmid pHMEF5-mCherry 19) were previously described.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis was performed as previously described. 18) Briefly, cell lysates were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGE), and transferred to polyvinyliden difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The HA-tagged Cpf1 proteins were detected using mouse Anti-HA antibody (Covance, Princeton, NJ, U.S.A.).…”

Section: Methodsmentioning

confidence: 99%

“…18) HEK293 cells were seeded on poly-L-lysine-coated 24-well plates (1×10 5 cells/well) and cotransfected with 400 ng of Cpf1-expressing plasmids, 400 ng of reporter pCAG-EGxxFP-AAVS1 and 200 ng of pHMEF5-mCherry as a transfection control using Lipofectamine 2000. Forty-eight hours after transfection, images were captured and processed using BIOREVO digital camera (BZ-9000, Keyence Japan, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The insetion/deletion (indel) mutations were assessed by T7E1 assay 2 and 14 d after transduction in H1299 cells and PHHs, respectively, as previously described. 18) Briefly, genomic DNA was prepared from the cells and the target region of human AAVS1 region was amplified by PCR using the primer set for AAVS1 (Table S1). PCRs were performed using PrimeSTAR Max DNA polymerase (TaKaRa Biomedicals, Otsu, Japan) following the manufacturer's instructions.…”

Section: T7 Endonuclease I (T7e1) Assaymentioning

confidence: 99%

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Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

Tsukamoto

Sakai

Iizuka

et al. 2018

Biological & Pharmaceutical Bulletin

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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system is now widely used as a genome editing tool. CRISPR-associated endonuclease in Prevotella and Francisella 1 (Cpf1) is a recently discovered Cas endonuclease that is designable and highly specific with efficiencies comparable to those of Cas9. Here we generated the adenovirus (Ad) vector carrying an Acidaminococcus sp. Cpf1 (AsCpf1) expression cassette (Ad-AsCpf1) for the first time. Ad-AsCpf1 was applied to primary human hepatocytes prepared from humanized mice with chimeric liver in combination with the Ad vector expressing the guide RNA (gRNA) directed to the Adeno-associated virus integration site 1 (AAVS1) region. The mutation rates were estimated by T7 endonuclease I assay around 12% of insertion/ deletion (indel). Furthermore, the transduced human hepatocytes were viable (ca. 60%) at two weeks post transduction. These observations suggest that the Ad vector-mediated delivery of the CRISPR/AsCpf1 system provides a useful tool for genome manipulation of human hepatocytes.

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Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

Ren

Peng

Ouyang

et al. 2018

Anim Models and Exp Med

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CRISPR /Cas9‐mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA . Unexpectedly, we found previously that pseudorabies virus ( PRV ) could escape from CRISPR /Cas9‐mediated inhibition. In order to elucidate whether the escape of PRV from Cas9‐mediated inhibition was due to cell deficiencies, such as genetic instability of sg RNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sg RNA ‐expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9‐mediated cleavage of PRV . Different passages of PX 459‐ PRV cells can stably express sg RNA to facilitate Cas9/sg RNA cleavage on the UL 30 gene of PRV , resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

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Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression

Cited by 7 publications

References 26 publications

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

Subculturing cells have no effect on CRISPR/Cas9‐mediated cleavage of UL30 gene in pseudorabies virus

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