CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.
AuthorsTsukamoto K, Ozeki C, Kohda T, Tsuji T,
Journal
PubMed ID26177297
'Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the ... More
Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.
AuthorsLiang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut JD,
Journal
PubMed ID26003884
'CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of ... More
Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.
AuthorsSakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T,
Journal
PubMed ID24954249
CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase ... More
Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.
AuthorsLiang X, Potter J, Kumar S, Ravinder N, Chesnut JD
JournalJ Biotechnol
PubMed ID27845164
'While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up ... More
Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.
AuthorsYu X, Liang X, Xie H, Kumar S, Ravinder N, Potter J, de Mollerat du Jeu X, Chesnut JD,
JournalBiotechnol Lett
PubMed ID26892225
'To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection. Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named ... More
Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC.
AuthorsPott LL, Hagemann S, Reis H, Lorenz K, Bracht T, Herold T, Skryabin BV, Megger DA, Kälsch J, Weber F, Sitek B, Baba HA
JournalOncotarget
PubMed ID28060762
Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. ... More
A functional screening of the kinome identifies the Polo-like kinase 4 as a potential therapeutic target for malignant rhabdoid tumors, and possibly, other embryonal tumors of the brain.
AuthorsSredni ST, Suzuki M, Yang JP, Topczewski J, Bailey AW, Gokirmak T, Gross JN, de Andrade A, Kondo A, Piper DR, Tomita T
JournalPediatr Blood Cancer
PubMed ID28398638
Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific ... More