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Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting (View Full Paper)

Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang & Gregory D. Davis
MilliporeSigma, 2909 Laclede Avenue, Saint Louis, Missouri 63103, USA
A Business of Merck KGaA, 64293 Darmstadt, Germany

 

 Abstract

Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

Chen, F. et al. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat. Commun. 8, 14958 doi: 10.1038/ncomms14958 (2017).

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Cat. No. Description
OLIGO Custom Oligo
I3390 Iscove’s modified Dulbecco’s medium
G7513 L-glutamine
D6046 Dulbecco’s modified Eagle’s medium
F2442 Fetal Bovine Serum
NA0410 GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit
D7440 JumpStart™ Taq ReadyMix™ for Quantitative PCR
RTN350 GenElute™ Mammalian Total RNA Miniprep Kit
E1014 Benzonase® Nuclease
T4455 TEV Protease
F3165 Monoclonal ANTI-FLAG® M2 antibody produced in mouse

 

 Infectious Disease – Zika Virus

Cell Host & Microbes, July 2016

A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection (View full paper)

Nicholas J. Barrows, Rafael K. Campos, Steven T. Powell, K. Reddisiva Prasanth, Geraldine Schott-Lerner, Ruben Soto-Acosta, Gaddiel Galarza-Muñoz, Erica L. McGrath, Rheanna Urrabaz-Garza, Junling Gao, Ping Wu, Ramkumar Menon, George Saade, Ildefonso Fernandez-Salas, Shannan L. Rossi, Nikos Vasilakis, Andrew Routh, Shelton S. Bradrick, Mariano A. Garcia-Blanco.
 

 Abstract

Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.
 

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Guava easyCyte Microcapillary Flow Cytometer Quantitation of infected cells See more publications of Guava
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