Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53

The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently emplo...

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Bibliographic Details
Published in:PloS one 2014-11, Vol.9 (11), p.e113232
Main Authors: Lawhorn, Ingrid E B, Ferreira, Joshua P, Wang, Clifford L
Format: Article
Language:English
Subjects:
Antigens
Bacteria
Biology and life sciences
Cell cycle
Cells (Biology)
Chemical engineering
Clustered Regularly Interspaced Short Palindromic Repeats - genetics
CRISPR
CRISPR-Associated Proteins - genetics
CRISPR-Associated Proteins - metabolism
Deoxyribonucleic acid
Design
DNA
Epigenetics
Gene expression
Gene silencing
Genes
Genetic Vectors - metabolism
Genomes
HEK293 Cells
Humans
Mammalian cells
Mutagenesis
Nuclease
p53 Protein
Proteins
Real-Time Polymerase Chain Reaction
Research and Analysis Methods
Researchers
RNA polymerase
RNA, Guide, CRISPR-Cas Systems - metabolism
RNA, Messenger - metabolism
Stem cells
Studies
Transcription factors
Transcription Initiation Site
Tumor proteins
Tumor Suppressor Protein p53 - genetics
Tumor Suppressor Protein p53 - metabolism
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Summary:The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0113232