Jan 6, 2016

CRISPR Technology for Genome Activation and Repression in Mammalian Cells

Cold Spring Harbor Protocols
Dan Du, Lei S Qi

Abstract

Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid underst...Continue Reading

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Mentioned in this Paper

Genome-Wide Association Study
Protein Complex Subunit Gene
HEK293 Cells
Genome
Genes
CRISPR-Cas Systems
CRISPR-Associated Protein Complex Proteins
Transcription, Genetic
Genomic Stability
DNA-like RNA

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