Tag: dCas9-vp64

LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2’-O-methylation

Ethics statement All mice procedures in this study were performed under an animal protocol approved by the Institutional Animal Care and Use Committee guidelines of Westlake University. The procedures and protocols for glioma patients were approved by the institutional review board of Beijing Tiantan Hospital. Informed consent was obtained from…

Continue Reading LncRNA INHEG promotes glioma stem cell maintenance and tumorigenicity through regulating rRNA 2’-O-methylation

IJMS | Free Full-Text | CRISPR/Cas9 Landscape: Current State and Future Perspectives

1. Introduction Genome editing has taken a leading position among genome modification technologies in a short time and is now widely used in gene therapy. To date, there are three main systems for genome editing: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas nucleases. Genome editing has…

Continue Reading IJMS | Free Full-Text | CRISPR/Cas9 Landscape: Current State and Future Perspectives

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

Modular iSBH-sgRNA designs enable spatial separation of spacer and trigger-sensing sequences. A. In second-generation iSBH-sgRNAs, RNA triggers are complementary with the iSBH-sgRNA backfolds, thus sgRNA spacers influence RNA trigger sequences. In modular iSBH-sgRNAs, design constrains were eliminated as triggers are only complementary with the iSBH-sgRNA loop and first 15nt of…

Continue Reading Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

IJMS | Free Full-Text | Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production

1. Introduction Peptides are employed for a multitude of therapeutic approaches and can be obtained from the native organ or tissue in which they are produced as well as through recombinant methods where nucleic acids encoding a candidate gene(s) are delivered to and expressed in cells in vitro. As an…

Continue Reading IJMS | Free Full-Text | Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Ethics statement All experiments in this study comply with guidelines of the U.S. Public Health Service and were approved by the Institutional Biosafety Committee at the University of Pittsburgh. All animal husbandry and experimental procedures were conducted in accordance with U.S. Public Health Service policy and approved by the Institutional…

Continue Reading Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

Cell culture We cultured mESCs in t2iL medium containing Dulbecco’s modified eagle medium (DMEM, Nacalai Tesque), 2 mM Glutamax (Nacalai Tesque), 1× non-essential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (P/S) (Nacalai Tesque), 0.1 mM 2-mercaptoethanol (Sigma) and 15% fetal bovine serum (FBS) (Gibco), supplemented with…

Continue Reading Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

SARS-CoV-2 restructures host chromatin architecture

Cell culture Human lung adenocarcinoma cells A549 expressing human ACE2 (A549-ACE2, NR-53821) were acquired from BEI Resources. They were maintained in DMEM/F-12 (1:1, Corning) medium supplemented with 10% FBS (GeneDepot) and blasticidin (100 μM). Normal A549 cells were purchased from ATCC (CCL-185) and cultured in DMEM/F-12 (1:1, Corning) supplemented with 10%…

Continue Reading SARS-CoV-2 restructures host chromatin architecture

CRISPRa experiment with a silent sgRNA at the LPL locus does not influence cell proliferation nor the expression of senescence marker genes.

(A) teloHAEC that express dCas9-VP64 were infected with a lentivirus that carries a sgRNA that targets rs1441755 at the LPL locus. This sgRNA was silent in all our pooled CRISPR screens for all six endothelial phenotypes tested. In the absence or presence of antibiotic selection (Zeocin), LPL_sg08237 does not affect…

Continue Reading CRISPRa experiment with a silent sgRNA at the LPL locus does not influence cell proliferation nor the expression of senescence marker genes.

ZFIN Publication: Weuring et al., 2021

PUBLICATION Authors Weuring, W.J., Dilevska, I., Hoekman, J., van de Vondervoort, J., Koetsier, M., van ‘t Slot, R.H., Braun, K.P.J., Koeleman, B.P.C. ID ZDB-PUB-210819-5 Date 2021 Source The CRISPR journal   4: 575-582 (Journal) Generate reference Registered Authors Koeleman, B. P. C., van ‘t Slot, Ruben, Weuring, Wout Keywords none…

Continue Reading ZFIN Publication: Weuring et al., 2021

Cellular glycan modification by B3GAT1 broadly restricts influenza virus infection

Ethics statement All procedures involving laboratory mice were approved by the Duke University IACUC under the protocol numbers A189-18-08 and A142-21-07. Mice were housed with up to five mice per cage and the ambient room conditions ranged from 70–74 °F and 30–70% humidity with a 12-hour dark/light cycle. Animals were assessed…

Continue Reading Cellular glycan modification by B3GAT1 broadly restricts influenza virus infection

dCas9-VP64-Blasticidin SAM CRISPRa Helper Construct 1 Plasmid DNA

This product is a lentiviral plasmid that utilizes the EF1 alpha promoter to drive expression of dCas9-VP64 and blasticidin resistance cassette linked by a 2A peptide (EF1a-dCas9-VP64-2A-Blasticidin) allowing for easy selection following successful transfection or transduction. Use Sigma′s lentiviral dCas9-VP64 plasmid for generation of lentiviral particles and efficient production of…

Continue Reading dCas9-VP64-Blasticidin SAM CRISPRa Helper Construct 1 Plasmid DNA