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Protocol

CRISPRmod dCas9-VPR or dCas9- SALL1-SDS3 mRNA and synthetic guide RNA transfection protocol

Clarence Mills, Horizon Team, Horizon/Perkin Elmer

Aug 10, 2021

Abstract

The following is a protocol for transfecting CRISPRmod dCas9-VPR or dCas9-SALL1-SDS3 mRNA with synthetic guide RNA into cultured mammalian cells using DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx). The protocol is written for transfection into 96-well tissue culture plates. 


Introduction

The CRISPR activation (CRISPRa) system is a variation of the canonical CRISPR-Cas9 used for creation of double-strand breaks in genomic DNA. It utilizes a nuclease-deactivated S. pyogenes Cas9 (dCas9), often called "dead Cas9", that is fused to one or more transcriptional activators. When paired with a well-designed guide RNA that targets a gene near a promoter region, the gene's native transcription start site is activated

dCas9-VPR mRNA expresses a human codon-optimized version of the nuclease-deactivated S. pyogenes Cas9 gene, the three transcriptional activators (VP64, p65 and Rta), and two nuclear localization signals (NLS). The dCas9-VPR mRNA is available in a form that co-expresses either EGFP or puromycin for transfection optimization or enrichment using fluorescence-activated cell sorting (FACS) or antibiotic selection.


Reagents and Equipment

DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx)


Procedure


Time Taken

1 hour

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