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 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.
Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
Ingrid E B
IE
Lawhorn
Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
Joshua P
JP
Ferreira
Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
Clifford L
CL
Wang
D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D005822:Genetic Vectors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D024363:Transcription Initiation Site; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53